Comparative proteomic analysis of sequential isolates of Mycobacterium tuberculosis from a patient with pulmonary tuberculosis turning from drug sensitive to multidrug resistant.

Background & objectives: Tuberculosis is a major health problem in India, and the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis (Mtb) has further complicated the situation. Though several studies characterizing drug sensitive and drug resistant strains are available in literature, almost all studies are done on unrelated strains. Therefore, the objective of this study was to compare the proteomic data of four sequential isolates of Mtb from a single patient who developed MDR-TB during the course of anti-tuberculosis therapy (ATT). Methods: In this study, using two-dimensional (2D) gel electrophoresis and MALDI-TOF mass spectrometry, we compared and analyzed the cell lysate proteins of Mtb sequential clinical isolates from a patient undergoing anti-TB treatment. The mRNA expression levels of selected identified proteins were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Results: The genotypes of all four isolates remained homologous, indicating no re-infection. The initial isolate (before treatment) was sensitive to all first-line drugs, but the consecutive isolates were found to be resistant to isoniazid (INH) and rifampicin (RIF) and developed mutations in the katG, inhA and rpoB. the intensities of 27 protein spots were found to be consistently overexpressed in INH and RIF resistant isolates. The most prominent and overexpressed proteins found during the development of drug resistance were GarA (Rv1827), wag31 (Rv2145c), Rv1437 and Rv2970c. Interpretation & conclusions: This preliminary proteomic study provides an insight about the proteins that are upregulated during drug resistance development. These upregulated proteins, identified here, could prove useful as immunodiagnostic and possibly drug resistant markers in future. However, more studies are required to confirm these findings.

Vitamin D levels in Indian children with intrathoracic tuberculosis.

Background & objectives: Deficiency of vitamin D, an immunomodulator agent, is associated with increased susceptibility to tuberculosis in adults, but only limited studies are available in the paediatric age group, especially regarding association of vitamin D with type and outcome of tuberculosis. We conducted this study to determine the baseline 25-hydroxy vitamin D levels in children suffering from intrathoracic tuberculosis and its association with type and outcome of tuberculosis. Methods: Children with intrathoracic tuberculosis, diagnosed on the basis of clinico-radiological criteria, were enrolled as part of a randomized controlled trial on micronutrient supplementation in paediatric tuberculosis patients. Levels of 25-hydroxy vitamin D were measured in serum samples collected prior to starting antitubercular therapy by chemiluminescent immunoassay technology. Results: Two hundred sixty six children (mean age of 106.9 ± 43.7 months; 57.1% girls) were enrolled. Chest X-ray was suggestive of primary pulmonary complex, progressive disease and pleural effusion in 81 (30.5%), 149 (56%) and 36 (13.5%) subjects, respectively. Median serum 25-hydroxy vitamin D level was 8 ng/ml (IQR 5, 12). One hundred and eighty six (69.9%) children were vitamin D deficient (serum 25-hydroxy vitamin D <12 ng/ml), 55 (20.7%) were insufficient (12 to <20 ng/ml) and 25 (9.4%) were vitamin D sufficient (≥ 20 ng/ml). lLevels of 25-hydroxy vitamin D were similar in all three types of intrathoracic tuberculosis, and in microbiologically confirmed and probable cases. Levels of 25-hydroxy vitamin D did not significantly affect outcome of the disease. Children who were deficient or insufficient were less likely to convert (become smear/culture negative) at two months as compared to those who were 25-hydroxy vitamin D sufficient (p<0.05). Interpretation & conclusions: Majority of Indian children with newly diagnosed intrathoracic tuberculosis were deficient in vitamin D. Type of disease or outcome was not affected by 25-hydroxy vitamin D levels in these children. However, children who did not demonstrate sputum conversion after intensive phase of antitubercular therapy had lower baseline 25-hydroxy vitamin D levels as compared to those who did.

Antibiotic susceptibilities, streptococcal pyrogenic exotoxin gene profiles among clinical isolates of group C or G Streptococcus dysgalactiae subsp. equisimilis & of group G S. anginosus group at a tertiary care centre.

Background & objectives: Group C and group G streptococci (together GCGS) are often regarded as commensal bacteria and their role in streptococcal disease burden is under-recognized. While reports of recovery of GCGS from normally sterile body sites are increasing, their resistance to macrolides, fluoroquinolone further warrants all invasive β haemolytic streptococci to be identified to the species level and accurately tested for antimicrobial susceptibility. This study was aimed to determine the prevalence, clinical profile, antimicrobial susceptibility and streptococcal pyrogenic exotoxin gene profile (speA, speB, speC, speF, smeZ, speI, speM, speG, speH and ssa) of GCGS obtained over a period of two years at a tertiary care centre from north India. Methods: The clinical samples were processed as per standard microbiological techniques. β-haemolytic streptococci (BHS) were characterized and grouped. Antimicrobial susceptibility of GCGS was performed using disk diffusion method. All GCGS were characterized for the presence of streptococcal pyrogenic exotoxins (spe) and spe genes were amplified by PCR method. Results: GCGS (23 GGS, 2GCS) comprised 16 per cent of β haemolytic streptococci (25/142 βHS, 16%) isolated over the study period. Of the 25 GCGS, 22 (88%) were recovered from pus, two (8%) from respiratory tract, whereas one isolate was recovered from blood of a fatal case of septicaemia. Of the total 23 GGS isolates, 18 (78%) were identified as Streptococcus dysgalactiae subsp equisimilis (SDSE, large-colony phenotype), five (21%) were Streptococcus anginosus group (SAG, small-colony phenotype). The two GCS were identified as SDSE. All GCGS isolates were susceptible to penicillin, vancomycin, and linezolid. Tetracycline resistance was noted in 50 per cent of SDSE isolates. The rates of macrolide and fluoroquinolone resistance in SDSE were low. Twelve of the 20 SDSE isolates were positive for one or more spe genes, with five of the SDSE isolates simultaneously carrying speA+ speB+ smeZ+ speF or speB+ smeZ+speF, speI+speM+speG+speH or, speI+spe M+speH or speA+ speB+ speC+ smeZ+ speF. One notable finding was the presence of spe B in four of the five isolates of the Streptococcus anginosus group. No isolate was positive for ssa. Interpretation & conclusions: Our study showed no association between GCGS isolates harbouring streptococcal pyrogenic exotoxins and disease severity. This might be attributed to the small sample size of spe-positive isolates.

Heat fixed but unstained slide smears are infectious to laboratory staff.

Background and Aim: In a clinical microbiology laboratory, heat fixed slide smears are commonly transported from one place to another for staining with different stains and also for onsite proficiency testing of laboratory technicians for accreditation of the laboratories. These smears are frequently handled without gloves by the staff in developing countries. Therefore, this study was conducted to check the survivability of tubercle bacilli on smears after physical and chemical treatments. Methods: A total of 196 AFB positive smears were analyzed. Of these, 116 were stained with Ziehl Neelsen (ZN), 60 with cold Kinyoun and 10 were unstained but heat fixed and 10 were neither stained nor heat fixed. The last 20 smears served as controls. The ZN and Kinyoun stained smears were 0-1.5-year-old and stored at room temperature in slide boxes, while control smears were freshly prepared. All smears were prepared from sputum samples positive for acid fast bacilli. All four sets were subjected to slide culture to see if mycobacteria could survive and grow in any. For slide culture, a new and safe device was used, which is designed for three in one purpose: cell cultivation, direct observation of the growth under microscope and cell harvesting inside the closed tube. The slide smears were directly dipped into this tube that contained liquid culture medium. The tubes were incubated at 370C for four weeks. The growth, if any, was confirmed by MPT-64 rapid test and subculture on LJ slants. Results: No growth was observed in ZN and Kinyoun stained slide smears. However, significant growth was observed in both control sets; the unstained non heat fixed as well as heat fixed slide smears. Conclusions: The results of our study indicate that tubercle bacilli remain viable even after heat fixation and carry risk of infection by contact. However, stained smears are safe for handling and storage.

Evaluation of toxicity & therapeutic efficacy of a new liposomal formulation of amphotericin B in a mouse model.

Background & objectives: Current therapy for leishmaniasis is limited and unsatisfactory. Amphotericin B, a second-line treatment is gradually replacing antimonials, the first-line treatment and is used as the preferred treatments in some regions. Though, presently it is the only drug with highest cure rate, its use is severely restricted by its acute toxicity. In the present study novel lipid-amphotericin B formulations with lower toxicity than the parent drug were evaluated for the treatment of visceral leishmaniasis (VL) in a mouse model. Methods: The toxicity and therapeutic efficacy of a new amphiphilic formulation of amphotericin B (KalsomeTM10) was compared to that of amphotericin B deoxycholate (Fungizone) in a mouse model of VL using quantitative real-time PCR (qRT-PCR). Results: The toxicity of amphotericin B was significantly less with liposomal formulation as compared to the deoxycholate form, evidenced by reduced nephrotoxicity and higher tolerated dose in BALB/c mice. The therapeutic efficacy was evaluated by quantitative real time (RT) PCR using primers highly specific for the ITS region of Leishmania donovani. There was reduction in parasite load by 2 log unit after 7 days of treatment and finally resulting in complete clearance of parasite from infected mice after 30 days of treatment with KalsomeTM10. Interpretation & conclusions: This new formulation showed a favourable safety profile and better efficacy when compared to conventional amphotericin B. If production cost is kept low, it may prove to be a feasible alternative to conventional amphotericin B.

Antiparasitic activity of plumericin & isoplumericin isolated from Plumeria bicolor against Leishmania donovani.

Background & objectives: The severe toxicity, exorbitant cost and emerging resistance of Leishmania species against most of the currently used drugs underscores the urgent need for the alternative drugs. The present study evaluates in vitro anti-leishmanial activity of Plumeria bicolor and its isolated compounds. Methods: The in vitro anti-parasitic activity of chloroform extract of Plumeria bicolor, plumericin and isoplumericin were tested alongwith appropriate controls against promastigote and amastigote forms of Leishmania donovani using 96 well microtiter plate. The concentration used for assessing the anti-leishmanial activity of extract of Plumeria bicolor and both isolated compounds were 100 μg/ml and 15 μM, respectively. The viability of the cells was assessed by MTT assay. The cytotoxicity of these compounds was performed against J774G8 murine macrophage cells lines at the concentration of 30 μM. Results: The Plumeria bicolor extract showed activity with the IC50 of 21±2.2 and 14±1.6 μg/ml against promastigote and amastigote forms of L. donovani, respectively. Plumericin consistently showed high activity with the IC50 of 3.17±0.12 and 1.41±0.03 μM whereas isoplumericin showed the IC50 of 7.2±0.08 μM and 4.1±0.02 μM against promastigote and amastigote forms, respectively. Cytotoxic effect of the chloroform extract of P. bicolor, plumericin and isoplumericin was evaluated in murine macrophage (J774G8) model with CC50 value of 75±5.3 μg/ml, 20.6±0.5 and 24±0.7 μM, respectively. Interpretation & conclusions: Our results indicated that plumericin showed more potent activity than isoplumericin and might be a promising anti-leishmanial agent against L. donovani.

Evaluation of risk factors for antituberculosis treatment induced hepatotoxicity.

Background & objectives: Antituberculosis (anti-TB) drug induced hepatotoxicity (DIH) is the most common side effect leading to interruption of therapy. Wide variations have been found in the reported incidence of hepatotoxicity during short-course chemotherapy. Several risk factors for hepatotoxicity have been suggested in previous studies. We undertook a prospective case-control study to assess the role of these putative risk factors in the development of DIH in patients receiving anti-TB treatment. Methods: One hundred and seventy five consecutive cases with a diagnosis of anti-TB DIH were compared with 428 consecutive controls who took anti-TB drugs for the full duration of chemotherapy without clinical or biochemical evidence of hepatitis. Cases positive for markers of acute viral hepatitis were carefully excluded. Cases and controls were compared with respect to age, sex, site of tuberculosis, radiological extent of disease on chest radiograph, body mass index (BMI), mid-arm circumference (MAC) and liver function at baseline which included serum bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), serum total protein and serum albumin. Results: Univariate logistic regression revealed that the risk of developing DIH was greater in older patients. Significantly greater percentage of cases had extrapulmonary tuberculosis (TB) (P<0.01). Also, a significantly higher percentage of cases had moderate to far advanced disease severity on chest radiograph (P<0.01). On multivariate logistic regression, the adjusted odds were significant (P<0.01) for age >35 yr, MAC <20 cm and hypoalbuminaemia (albumin <3.5 g/dl). Interpretation & conclusions: Older age, poor nutritional status including baseline hypoalbuminaemia were independent predictors of occurrence of anti-TB DIH. Clinicians should be vigilant for occurrence of hepatotoxicity in this high risk group.

Low serum zinc levels in an endemic area of visceral leishmaniasis in Bihar, India.

Background & objectives: India carries approximately 50 per cent of the global burden of visceral leishmaniasis and majority of patients from the poor, rural communities of Bihar State. Zinc is an essential trace element and its relevance for proper functioning of the entire immune system is already well documented. Though low serum zinc levels have been reported in many parasitic diseases, limited information is available regarding zinc status in human leishmaniasis. We investigated to define the relationship between zinc level in visceral leishmaniasis (VL) patients in endemic and non-endemic regions. Methods: Venous blood was collected from 88 patients, 16 parasitologically confirmed VL, 35 healthy controls from endemic area (Bihar) and 37 healthy urban controls from non-endemic area, Delhi. In all the three groups, levels of serum albumin, total protein (markers of nutritional status) and zinc were estimated by colorimetric methods. Results: Serum zinc levels were found to be significantly lower (P<0.001) in VL patients than non-endemic controls. The serum zinc levels in VL endemic controls were also significantly lower (P<0.001) than non-endemic controls, but these values were not statistically significantly different from VL patients. However, all samples from Bihar (VL patients and controls) had lower serum zinc levels than non-endemic controls from Delhi. Interpretation & conclusion: Low serum Zn levels, in healthy subjects from Bihar and more significantly in VL patients of this region, are possibly associated with vulnerability and endemicity of visceral leishmaniasis in the region. Further studies need to be done to assess the role of oral zinc supplementation in better management and prevention of VL, particularly in endemic areas.

Proteomic analysis of heparin-binding proteins from human seminal plasma: a step towards identifi cation of molecular markers of male fertility.

Glycosaminoglycans, especially heparin, are involved in various cell processes such as apoptosis, cell cycle control, platelet activation, capacitation, acrosome reaction and sperm decondensation. Heparin-binding proteins (HBPs) are essential constituents of human seminal fl uid, which bind to sperm lipids containing the phosphorylcholine group and mediate the fertilization process. We utilized a proteomic set-up consisting of affi nity chromatography, isoelectric focusing (IEF) coupled with matrix-assisted laser desorption/ionization time-of-fl ight tandem mass spectrometry (MALDI TOF/MS) for protein analysis of human HBPs. We resolved 70 different spots on two-dimensional (2-D) gel and subsequently identifi ed these proteins. Forty different types of proteins were identifi ed. Functional analysis revealed that 38% of the proteins belonged to the enzyme category, 20% were involved in RNA processing and transcription, 18% in structure and transport function, and 16% in cell recognition and signal transduction. We also identifi ed 8% of proteins with unknown functions, although their expression in seminal fl uid has been documented. Proteins of seminal fl uid that bind heparin may be directly involved in sperm capacitation and acrosome reaction (AR), which are the two critical steps for fertilization. This information on HBPs would be useful for identifying potential biomarkers of fertility in the near future.

Acute bilateral vision loss as the presenting feature of subacute sclerosing panencephalitis.

A 12-yr-old boy with an atypical presentation of subacute sclerosing panencephalitis (SSPE) is described. Bilateral macular chorioretinitis preceded the neurological symptoms by 3 weeks. Both visual and neurological features had an acute onset. Clinicians need to be aware that macular chorioretinitis in a child may be the heralding feature of SSPE.

Role of acute viral hepatitis as a confounding factor in antituberculosis treatment induced hepatotoxicity.

BACKGROUND & OBJECTIVE: Drug induced hepatotoxicity (DIH) is an important and commonly encountered adverse effect with antituberculosis (anti-TB) treatment. Acute viral hepatitis (AVH) is an important confounding reason which clinically, biochemically and histologically mimics DIH. METHODS: The contributory role of acute viral hepatitis as a confounding factor in patients with normal baseline liver functions who developed acute hepatitis while receiving short-course anti-TB treatment was prospectively studied. The sera of all patients who developed acute hepatitis were analysed for markers for hepatitis A, B, C and E viruses. RESULTS: Viral hepatitis was present in 15 of the 102 (14.7%) patients who developed acute hepatitis while receiving anti-TB treatment with hepatitis E virus being the most common cause Later onset of acute hepatitis [58 (5-133) vs. 26 (3-221) days; P=0.04], large elevations in aspartate aminotransferase (AST) [371 (30-2643) vs. 212 (63-1990 IU/l); P=0.03] and alanine aminotransferase (ALT) [388 (31-2997) vs. 225 (52- 1670 IU/l); P= 0.002] and a longer time for normalization of deranged liver functions [36.7 +/- 13.3 vs. 24.5 +/- 19.3 days; P=0.02] indicated acute viral hepatitis as the cause of liver function derangement. INTERPRETATION & CONCLUSION: Our findings showed AVH in 14.7 per cent patients who developed hepatotoxicity while an anti-TB treatment. Therefore, in endemic areas, viral hepatitis should be sought after and excluded in all patients suspected to have DIH before attributing the hepatotoxic effect to the anti-TB drugs.

Insect vectors of Leishmania: distribution, physiology and their control.

Leishmaniasis is a deadly vector-borne disease that causes significant morbidity and mortality in Africa, Asia, Latin America and Mediterranean regions. The causative agent of leishmaniasis is transmitted from man to man by a tiny insect called sandfly. Approximately, 600 species of sandflies are known but only 10% of these act as disease vectors. Further, only 30 species of these are important from public health point. Fauna of Indian sub-zone is represented by 46 species, of these, 11 belong to Phlebotomine species and 35 to Sergentomyia species. Phlebotomus argentipes is the proven vector of kala-azar or visceral leishmaniasis in India. This review gives an insight into the insect vectors of human leishmaniasis, their geographical distribution, recent taxonomic classification, habitat, and different control measures including indoor residual spraying (IRS), insecticide-treated bednets (ITNs), environmental management, biological control, and emerging resistance to DDT. Role of satellite remote sensing for early prediction of the disease by identifying the sandflygenic conditions cannot be undermined. The article also underlines the importance of synthetic pheromones which can be used in near future for the control of these vectors.

New developments in diagnosis of leishmaniasis.

In this review recent advances made in the field of human leishmaniasis have been discussed with special emphasis on the parasite, and various serological and molecular methods of diagnosing the infection. The article also reviews various modes of parasite transmission including vector borne, blood transfusion, needle sharing, sexual and person-to-person. Microbiological methods including the bone marrow, spleen, liver, lymph node aspirations and various staining methods used to demonstrate the amastigotes of the parasites and various in vitro promastigote culture methods are discussed in detail with their comparative sensitivity rates.

Mother-to-child transmission of hepatitis E virus infection.

OBJECTIVES: Water borne or enterically transmitted non-A-non-B hepatitis is a major public health problem in India. Many of these cases carry fatal outcome. The hepatitis E virus (HEV) has been considered to be the most important causative agent of this entity. The severity and fatality rates of HEV infection are reported to be rather more in pregnant women. However, there is meager information from India, on mother to child transmission of this agent. METHODS: During 1997-98, we studied 60 pregnant women suspected to have acute viral hepatitis to understand the frequency of various viral etiologies, disease course and outcome of the pregnancy. Six cord blood samples were tested for IgG, and IgM antibodies against hepatropic viral agents and also for hepatitis E virus RNA by RT-nested PCR using ORF-1 as target. RESULTS: Of the 60 pregnant patients hospitalised at All India Institute of Medical Sciences, New Delhi for acute hepatitis, 22 (37%) were positive for IgM anti-HEV antibodies and 10% were infected with hepatitis B virus. Co-infection of HEV with Hepatitis B and C was seen in 1 and 2 patents, respectively. Most (72%) of the HEV infected patients were in third trimester of pregnancy (P<0.05). Of the 6 cord blood samples tested 3 (50%) were positive for HEV RNA. Though, all mothers were RNA positive, half of the babies did not get infected in utero with HEV. Fourteen of the 22 (63.6%) HEV infected mothers developed fulminant hepatic failure and all died. CONCLUSION: The mortality rate in HEV [corrected] infected mothers was 100%. Mother to child transmission of hepatitis E virus infection was established in 50%.