Biomedical subjects

Sanjeev Krishna

Publications and source records attributed to Sanjeev Krishna.

At least 19 recordsLinked to original sources

PubMed2005

Amplification of Plasmodium falciparum multidrug resistance gene 1 in isolates from Gabon.

The study of molecular markers of drug resistance is particularly important in surveillance studies of drugs, such as mefloquine, that still retain efficacy in sub-Saharan Africa yet have encountered resistance elsewhere. In a recent study in Thailand, we identified amplification of the Plasmodium falciparum multidrug resistance gene 1 (pfmdr1) as being the most important predictor of in vitro drug resistance and in vivo failure of mefloquine monotherapy. Here we report amplification of pfmdr1 in >5% of patient samples from Lambarene, Gabon, collected in 1995. None of the samples collected 7 years later showed pfmdr1 amplification, suggesting that parasites with increased numbers of pfmdr1 copies have not substantially spread through the population. Nevertheless, the detection of multicopy pfmdr1 in African parasites suggests a high potential for rapid selection for resistance, implying that mefloquine use in Africa should be considered only as part of combination therapy.

ATP-Binding Cassette Transporters↗

PubMed2005

The prognostic value of measures of acid/base balance in pediatric falciparum malaria, compared with other clinical and laboratory parameters.

BACKGROUND: Identifying severe, life-threatening falciparum malaria in African children allows for the prompt institution of appropriate management. In the past 2 decades, hyperlactatemia and acidosis have been identified as being associated with mortality in patients with severe malaria, but measurement of blood lactate concentration and base excess is expensive and technically demanding. In this large, prospective study, we examined the prognostic value of acidosis and hyperlactatemia and compared these markers to clinically assessed variables. METHODS: We examined several clinical and laboratory measurements as prognostic markers of mortality in 14,605 parasitemic children admitted to 3 hospitals in Africa. Whole-blood lactate concentration and acid/base status were used to identify subjects who had hyperlactatemia and acidosis. RESULTS: Using cut-points established by sensitivity and specificity curves, the sensitivities and positive predictive values for both lactate concentration and base excess were low, the specificities were moderate, and the negative predictive values were high (>97%). No reliable clinical surrogates for hyperlactatemia or acidosis were identified. Addition of lactate concentration and base excess to predictive models with previously identified clinical features (Blantyre Coma Score, deep breathing, prostration, and weight-for-age Z score) and 1 laboratory measure (blood glucose level) did not appreciably improve models to predict mortality. CONCLUSIONS: Measurements of lactate concentration and acid/base balance are expensive to perform, and performance of the latter can be problematic. Severe falciparum malaria may be readily recognized in children at admission to hospitals in sub-Saharan Africa with use of simple, inexpensive means and does not require knowledge of lactate concentration and base excess.

Acid-Base Equilibrium↗

PubMed2005

A single amino acid residue can determine the sensitivity of SERCAs to artemisinins.

Artemisinins are the most important class of antimalarial drugs. They specifically inhibit PfATP6, a SERCA-type ATPase of Plasmodium falciparum. Here we show that a single amino acid in transmembrane segment 3 of SERCAs can determine susceptibility to artemisinin. An L263E replacement of a malarial by a mammalian residue abolishes inhibition by artemisinins. Introducing residues found in other Plasmodium spp. also modulates artemisinin sensitivity, suggesting that artemisinins interact with the thapsigargin-binding cleft of susceptible SERCAs.

Amino Acid Sequence↗

PubMed2005

Artesunate-clindamycin versus quinine-clindamycin in the treatment of Plasmodium falciparum malaria: a randomized controlled trial.

BACKGROUND: Artemisinin-based drug combinations are the mainstay in the fight against drug-resistant malaria in Africa. Currently available antimalarial drug combinations that include artemisinins are pharmacokinetically unmatched and are therefore potentially increasing the risk of selection of resistant mutants in areas in which the rate of transmission of malaria is high. We tested the potential value of artemisinin-based combination therapy with a short elimination half-life for the treatment of uncomplicated Plasmodium falciparum malaria in sub-Saharan Africa. METHODS: We conducted an open-label, randomized, controlled clinical trial to evaluate the efficacy and tolerability of oral artesunate-clindamycin therapy given twice daily for 3 days (artesunate, 2 mg/kg, and clindamycin, 7 mg/kg, per dose), compared with a standard quinine-clindamycin regimen given twice daily for 3 days (quinine, 15 mg/kg, and clindamycin, 7 mg/kg, per dose), for the treatment of uncomplicated falciparum malaria in 100 Gabonese children aged 3-12 years. The primary end point of the study was the polymerase chain reaction-corrected cure rate for the per-protocol population. RESULTS: The activity of artesunate-clindamycin was comparable to that of quinine-clindamycin in the per-protocol analysis of cure rates at day 28 of follow-up (87% versus 94%). No serious adverse events were reported, and tolerability was good and was similar in both groups. Times to clearance of fever and clearance of parasites were significantly shorter in the artesunate-clindamycin group. CONCLUSIONS: Artesunate-clindamycin and other matching artemisinin-based combinations with a short plasma half-life merit further attention for use in regions in which the rate of transmission of malaria is high.

Antimalarials↗

PubMed2005

Severe falciparum malaria in Gabonese children: clinical and laboratory features.

BACKGROUND: Malaria continues to claim one to two million lives a year, mainly those of children in sub-Saharan Africa. Reduction in mortality depends, in part, on improving the quality of hospital care, the training of healthcare workers and improvements in public health. This study examined the prognostic indicators of severe falciparum malaria in Gabonese children. METHODS: An observational study examining the clinical presentations and laboratory features of severe malaria was conducted at the Centre Hospitalier de Libreville, Gabon over two years. Febrile children aged from 0 to 10 years with Plasmodium falciparum infection and one or more features of severe malaria were enrolled. RESULTS: Most children presenting with severe falciparum malaria were less than 5 years (92.3% of 583 cases). Anaemia was the most frequent feature of severe malaria (67.8% of cases), followed by respiratory distress (31%), cerebral malaria (24%) hyperlactataemia (16%) and then hypoglycaemia (10%). Anaemia was more common in children under 18 months old, while cerebral malaria usually occurred in those over 18 months. The overall case fatality rate was 9%. The prognostic indicators with the highest case fatality rates were coma/seizures, hyperlactataemia and hypoglycaemia, and the highest case fatality rate was in children with all three of these features. CONCLUSIONS: Prompt and appropriate, classification and treatment of malaria helps identify the most severely ill children and aids early and appropriate management of the severely ill child.

Age Distribution↗

PubMed2005

The Nramp orthologue of Cryptococcus neoformans is a pH-dependent transporter of manganese, iron, cobalt and nickel.

Cryptococcus neoformans is an important human opportunistic pathogen and a facultative intracellular parasite, particularly in HIV-infected individuals. Little is known about metal ion transport in this organism. C. neoformans encodes a single member of the Nramp (natural resistance-associated macrophage protein) family of bivalent cation transporters, known as Cramp, which we have cloned and expressed in Xenopus laevis oocytes and Spodoptera frugiperda Sf 21 insect cells. Cramp induces saturable transport of a broad range of bivalent transition series cations, including Mn2+, Fe2+, Co2+ and Ni2+. Maximal cation transport occurs at pH 5.5-6.0, consistent with the proton gradient-based energetics of other Nramp orthologues. Mn2+ transport is diminished in the presence of 140 mM Na+, compatible with a Na+ slippage mechanism proposed for the Saccharomyces cerevisiae Nramp orthologue Smf1p. Cramp resembles Smf1p with respect to predicted membrane topology, substrate specificity and pH dependence, but differs in terms of its apparent affinity for Mn2+ and negligible inhibition by Zn2+. Cramp is the first Nramp orthologue from a fungal pathogen to be functionally characterized. Insights afforded by these findings will allow the formulation of new hypotheses regarding the role of metal ions in the pathophysiology of cryptococcosis.

Animals↗

PubMed2005

Proteomic fingerprinting for the diagnosis of human African trypanosomiasis.

Papadopoulos et al. recently reported the discovery of a diagnostic serum proteomic signature for human African trypanosomiasis (HAT), using a combination of surface-enhanced laser desorption-ionization time-of-flight (SELDI-TOF) mass spectrometry and data-mining algorithms. This novel approach, coupled with biochemical characterization of the proteins that contribute to the signature, provides powerful new tools for the development of improved diagnostic tests, disease staging and identification of potential novel drug targets in HAT.

Algorithms↗

PubMed2005

The relevance of malaria pathophysiology to strategies of clinical management.

PURPOSE OF REVIEW: Malaria claims 1-2 million lives a year, mostly children in sub-Saharan Africa. The majority of hospital deaths occur within 24 h of admission despite adequate treatment with antimalarial chemotherapy. Understanding the pathophysiological disturbances of malaria should allow the development of supportive therapy to "buy time" for antimalarial chemotherapy to clear the infection. It is sobering, however, that despite many trials over the last quarter of a century all large trials of adjunctive therapy so far have resulted in either increased morbidity or mortality, or both. RECENT FINDINGS: Severe malaria may be divided broadly into neurological and metabolic complications. We review recent findings about the pathophysiology of these complications and the implications for future adjunctive therapy of malaria, including the proposed importance of fluid volume depletion and sequestration of parasitized red cells in severe malaria. We also consider other anaemia, hyperparasitaemia and renal failure, which also require urgent treatment in severe malaria. SUMMARY: We review the important pathophysiological features of severe malaria and promising adjunctive therapies such as dichloroacetate that warrant further larger trials to determine whether they improve the so-far intractable death rate of severe malaria.

Acidosis, Lactic↗

PubMed2004

Assessment of volume depletion in children with malaria.

BACKGROUND: The degree of volume depletion in severe malaria is currently unknown, although knowledge of fluid compartment volumes can guide therapy. To assist management of severely ill children, and to test the hypothesis that volume changes in fluid compartments reflect disease severity, we measured body compartment volumes in Gabonese children with malaria. METHODS AND FINDINGS: Total body water volume (TBW) and extracellular water volume (ECW) were estimated in children with severe or moderate malaria and in convalescence by tracer dilution with heavy water and bromide, respectively. Intracellular water volume (ICW) was derived from these parameters. Bioelectrical impedance analysis estimates of TBW and ECW were calibrated against dilution methods, and bioelectrical impedance analysis measurements were taken daily until discharge. Sixteen children had severe and 19 moderate malaria. Severe childhood malaria was associated with depletion of TBW (mean [SD] of 37 [33] ml/kg, or 6.7% [6.0%]) relative to measurement at discharge. This is defined as mild dehydration in other conditions. ECW measurements were normal on admission in children with severe malaria and did not rise in the first few days of admission. Volumes in different compartments (TBW, ECW, and ICW) were not related to hyperlactataemia or other clinical and laboratory markers of disease severity. Moderate malaria was not associated with a depletion of TBW. CONCLUSIONS: Significant hypovolaemia does not exacerbate complications of severe or moderate malaria. As rapid rehydration of children with malaria may have risks, we suggest that fluid replacement regimens should aim to correct fluid losses over 12-24 h.

Child↗

PubMed2004

Metal ion transport and regulation in Mycobacterium tuberculosis.

The regulation of metal ion concentrations is central to the physiology of the interaction between pathogenic bacteria and their hosts. Apart from the NRAMP orthologue, MntH, metal ion transporters in Mycobacterium tuberculosis have not been studied. Mn, the physiological substrate of MntH in other bacteria, may play an important role as a structural and redox-active cofactor in a wide range of metabolic processes. Fe, Cu and Zn play structural and catalytic roles in metalloenzymes involved in oxidative stress responses. Fe and Mg are required for growth in macrophages. Genomic analyses reveal 28 sequences encoding a broad repertoire of putative metal ion transporters (or transporter subunits), representing 24% of all transporters in this organism. These comprise 8 families of secondary active transporters and 3 families of primary active transporters, including 12,P, type ATPases. Potential metal ion specificities include K+, Na+, Cu2+, Cd2+, Zn2+, Mn2+, Mg2+, Ca2+, Co2+, Ni2+, Fe2+/3+, Hg2+, AsO2- and AsO4(2-). 17 of these transporters are also encoded as complete open reading frames in Mycobacterium leprae, suggesting a role in intracellular survival. Iron transcriptionally regulates a diverse set of genes via the iron-dependent DNA-binding proteins, Fur and IdeR. Changes in Fe and Mg concentrations signal entry into the intracellular compartment and potentially trigger up-regulation of virulence determinants. The plethora of putative transport systems encoded by the M. tuberculosis genome contrasts strikingly with the paucity of experimental data on these systems. The detailed analysis of the temporal pattern of M. tuberculosis transporter gene expression during infection will provide important insights into the basic biology of intracellular parasitism and may help to shape novel therapeutic strategies.

Adenosine Triphosphatases↗

PubMed2004

Analysis of Plasmodium vivax hexose transporters and effects of a parasitocidal inhibitor.

Plasmodium vivax is the second most common species of malaria parasite and causes up to 80 million episodes of infection each year. New drug targets are urgently needed because of emerging resistance to current treatments. To study new potential targets, we have functionally characterized two natural variants of the hexose transporter of P. vivax (PvHT) after heterologous expression in Xenopus oocytes. We show that PvHT transports both glucose and fructose. Differences in the affinity for fructose between the two variants of PvHT establishes that sequence variation is associated with phenotypic plasticity. Mutation of a single glutamine residue, Gln(167), predicted to lie in transmembrane helix 5, abolishes fructose transport by PvHT, although glucose uptake is preserved. In contrast, the exofacial site located between predicted helices 5 and 6 of PvHT is not an important determinant of substrate specificity, despite exhibiting sequence polymorphisms between hexose transporters of different Plasmodium spp. Indeed, replacement of twelve residues located within this region of PvHT by those found in the orthologous Plasmodium falciparum sequence (PfHT) is functionally silent with respect to affinity for hexoses. All PvHT variants are inhibited by compound 3361, a long-chain O-3 derivative of D-glucose effective against PfHT. Furthermore, compound 3361 kills short term cultures of P. vivax isolated from patients. These data provide unique insights into the function of hexose transporters of Plasmodium spp. as well as further evidence that they could be targeted by drugs.

Amino Acid Sequence↗

PubMed2004

Inhibition of hexose transport and abrogation of pH homeostasis in the intraerythrocytic malaria parasite by an O-3-hexose derivative.

An O-3-hexose derivative, shown previously to inhibit a malaria parasite hexose transporter expressed in Xenopus oocytes as well as to suppress the multiplication of parasites, both in vitro and in vivo, was shown here to block the uptake of hexose sugars into isolated blood-stage parasites. This led to a decline in ATP levels and the loss of intracellular pH control. The results are consistent with those obtained with the cloned transporter. They support the notion that the transporter mediates uptake of glucose into the intraerythrocytic parasite and provide further support for the view that it is a suitable antimalarial drug target.

Adenosine Triphosphatases↗

PubMed2004

Aquaporin-4 facilitates reabsorption of excess fluid in vasogenic brain edema.

Aquaporin-4 (AQP4) is the major water channel in the brain, expressed predominantly in astroglial cell membranes. Initial studies in AQP4-deficient mice showed reduced cellular brain edema following water intoxication and ischemic stroke. We hypothesized that AQP4 deletion would have the opposite effect (increased brain swelling) in vasogenic (noncellular) edema because of impaired removal of excess brain water through glial limitans and ependymal barriers. In support of this hypothesis, we found higher intracranial pressure (ICP, 52+/-6 vs. 26+/-3 cm H2O) and brain water content (81.2+/-0.1 vs. 80.4+/-0.1%) in AQP4-deficient mice after continuous intraparenchymal fluid infusion. In a freeze-injury model of vasogenic brain edema, AQP4-deficient mice had remarkably worse clinical outcome, higher ICP (22+/-4 vs. 9+/-1 cm H2O), and greater brain water content (80.9+/-0.1 vs. 79.4+/-0.1%). In a brain tumor edema model involving stereotactic implantation of melanoma cells, tumor growth was comparable in wild-type and AQP4-deficient mice. However, AQP4-deficient mice had higher ICP (39+/-4 vs. 19+/-5 cm H2O at seven days postimplantation) and corresponding accelerated neurological deterioration. Thus, AQP4-mediated transcellular water movement is crucial for fluid clearance in vasogenic brain edema, suggesting AQP4 activation and/or up-regulation as a novel therapeutic option in vasogenic brain edema.

Animals↗

PubMed2004

A novel and accurate diagnostic test for human African trypanosomiasis.

INTRODUCTION: Human African trypanosomiasis (sleeping sickness) affects up to half a million people every year in sub-Saharan Africa. Because current diagnostic tests for the disease have low accuracy, we sought to develop a novel test that can diagnose human African trypanosomiasis with high sensitivity and specificity. METHODS: We applied serum samples from 85 patients with African trypanosomiasis and 146 control patients who had other parasitic and non-parasitic infections to a weak cation exchange chip, and analysed with surface-enhanced laser desorption-ionisation time-of-flight mass spectrometry. Mass spectra were then assessed with three powerful data-mining tools: a tree classifier, a neural network, and a genetic algorithm. FINDINGS: Spectra (2-100 kDa) were grouped into training (n=122) and testing (n=109) sets. The training set enabled data-mining software to identify distinct serum proteomic signatures characteristic of human African trypanosomiasis among 206 protein clusters. Sensitivity and specificity, determined with the testing set, were 100% and 98.6%, respectively, when the majority opinion of the three algorithms was considered. This novel approach is much more accurate than any other diagnostic test. INTERPRETATION: Our report of the accurate diagnosis of an infection by use of proteomic signature analysis could form the basis for diagnostic tests for the disease, monitoring of response to treatment, and for improving the accuracy of patient recruitment in large-scale epidemiological studies.

Adolescent↗

PubMed2004

The hexose transporter of Plasmodium falciparum is a worthy drug target.

Despite substantial efforts at control over several decades, malaria is still a major global health problem as chemotherapy of malaria parasites is limited by established drug resistance and lack of novel treatment options. Intraerythrocytic stages of these parasites are wholly dependent upon host glucose for energy and malarial proteins involved in hexose permeation are therefore attractive new drug targets. For Plasmodium falciparum, the causative agent of severe malaria, a facilitative hexose transporter (PfHT), encoded by a single-copy gene mediates glucose uptake. We first established heterologous expression in Xenopus laevis to allow functional characterisation of PfHT. This review describes the value of using Xenopus oocytes in heterologous studies of P. falciparum-encoded proteins and summarises the properties of PfHT. Comparisons between Gluts (mammalian facilitative hexose transporters) and PfHT using this expression system have highlighted important mechanistic and structural differences between parasite and host proteins. Certain O-methyl derivatives of glucose proved particularly useful discriminators between mammalian transporters and PfHT. We exploited this selectivity and synthesised a long-chain O-3-hexose derivative (compound 3361) that potently inhibits PfHT expressed in oocytes and also kills P. falciparum when it is cultured in medium containing either glucose or fructose as a carbon source. To extend our observations to the second most important human malarial pathogen, we have cloned and expressed the Plasmodium vivax orthologue of PfHT, and demonstrate inhibition of glucose uptake by compound 3361. These findings validate malarial hexose transporters as a novel target. We now aim to design a new class of antimalarials by the discovery of highly specific inhibitors which could act with a broad spectrum of action on different Plasmodium spp. infections.

Animals↗

PubMed2004

Artemisinins: activities and actions.

Multidrug-resistant malaria caused by Plasmodium falciparum has severely limited treatment options over recent years. Artemisinins are still effective for treating uncomplicated as well as severe malaria, because resistance is not yet clinically apparent. This article reviews some clinically useful properties of artemisinins and how they might work.

Animals↗

PubMed2004

Retaking sleeping sickness control in Angola.

Africa is severely affected by a resurgence of human African trypanosomiasis (HAT) at epidemic proportions. We report the results of the first 5 years of a HAT control programme in northern Angola run by the non-governmental organization (NGO) ANGOTRIP. In the period between 1996 and 2001, 13 426 patients were screened for HAT. The mortality rate of patients in stage II who were treated with melarsoprol fell from 7.5% to 2.9%, possibly as a result of training and the standardization of treatment protocols. A total of 191,578 people in three provinces of Angola were screened for HAT. Vector control activities were initiated using Lancien traps. Our experiences reflect the connection between war and the increasing incidence of disease, but also demonstrate that HAT control is possible by dedicated NGOs in close cooperation with national institutions even under extremely difficult circumstances.

Adolescent↗

PubMed2004

Short report: evaluation of a simple and inexpensive photometric device for the measurement of hemoglobin.

We have evaluated the accuracy of a simple and inexpensive photometric device (DHT) for the estimation of the blood concentration of hemoglobin by comparison with an automated, high-resolution, flow cytometry-based hematology analyzer (CellDyn 3000) and a centrifugal quantitative buffy coat hematology system (QBC I). We have analyzed the hemoglobin values of 163 individual blood samples. Bland-Altman analysis showed that the methods agreed only poorly: mean differences were 1.0 g/dL with limits of agreement (LOA) of -1.2 g/dL to 3.2 g/dL for the comparison of DHT and CellDyn measurements, 0.5 g/dL with LOA of -2.0 g/dL to 3.0 g/dL for the comparison of DHT with QBC measurements, and 0.5 g/dL with LOA of -1.1 g/dL to 2.1 g/dL for the comparison of QBC with CellDyn measurements. We conclude that the poor agreement of the DHT with the CellDyn does not permit the use of the DHT for critical hemoglobin measurements, particularly in transfusion services.

Colorimetry↗