Characterisation of complexes formed by parasite proteins exported into the host cell compartment of Plasmodium falciparum infected red blood cells.

During its intraerythrocytic life cycle, the human malaria parasite Plasmodium falciparum supplements its nutritional requirements by scavenging substrates from the plasma through the new permeability pathways (NPPs) installed in the red blood cell (RBC) membrane. Parasite proteins of the RhopH complex: CLAG3, RhopH2, RhopH3, have been implicated in NPP activity. Here, we studied 13 exported proteins previously hypothesised to interact with RhopH2, to study their potential contribution to the function of NPPs. NPP activity assays revealed that the 13 proteins do not appear to be individually important for NPP function, as conditional knockdown of these proteins had no effect on sorbitol uptake. Intriguingly, reciprocal immunoprecipitation assays showed that five of the 13 proteins interact with all members of the RhopH complex, with PF3D7_1401200 showing the strongest association. Mass spectrometry-based proteomics further identified new protein complexes; a cytoskeletal complex and a Maurer's clefts/J-dot complex, which overall helps clarify protein-protein interactions within the infected RBC (iRBC) and is suggestive of the potential trafficking route of the RhopH complex itself to the RBC membrane.

Anti-Helicobacter pylori activity of ethoxzolamide.

Ethoxzolamide (EZA), acetazolamide, and methazolamide are clinically used sulphonamide drugs designed to treat non-bacteria-related illnesses (e.g. glaucoma), but they also show antimicrobial activity against the gastric pathogen Helicobacter pylori. EZA showed the highest activity, and was effective against clinical isolates resistant to metronidazole, clarithromycin, and/or amoxicillin, suggesting that EZA kills H. pylori via mechanisms different from that of these antibiotics. The frequency of single-step spontaneous resistance acquisition by H. pylori was less than 5 × 10-9, showing that resistance to EZA does not develop easily. Resistance was associated with mutations in three genes, including the one that encodes undecaprenyl pyrophosphate synthase, a known target of sulphonamides. The data indicate that EZA impacts multiple targets in killing H. pylori. Our findings suggest that developing the approved anti-glaucoma drug EZA into a more effective anti-H. pylori agent may offer a faster and cost-effective route towards new antimicrobials with a novel mechanism of action.

Group B Streptococcus among Pregnant Women and Newborns in Mirzapur, Bangladesh: Colonization, Vertical Transmission, and Serotype Distribution.

Group B streptococcus (GBS) infection is a leading cause of death among newborns in developed countries. Data on the burden of GBS in Asian countries are lacking. This study aimed to understand (i) the rate of maternal rectovaginal GBS carriage, (ii) the rate of vertical transmission of GBS, as determined by culturing ear, umbilicus, and nasal swabs, and (iii) the distribution of GBS serotypes. This prospective observational study was conducted between September 2012 and November 2013 at Kumudini Women's Medical College Hospital, a secondary-level hospital in Mirzapur, Bangladesh. The study enrolled pregnant women who visited the outpatient clinic for antenatal care (ANC) and/or delivered a child in the inpatient department of Kumudini Women's Medical College Hospital and the babies born to those mothers. Among 1,151 enrolled pregnant women, 172 (15% [95% confidence interval [CI], 13 to 17%]) carried GBS; among 68 babies born to mothers with carriage, 26 (38% [95% CI, 27 to 51%]) had GBS on their body surfaces, indicating vertical transmission. Typing of the isolates (n = 172) identified all 10 GBS serotypes, most commonly types Ia (40% [69/172 isolates]), V (23% [40/172 isolates]), II (14% [24/172 isolates]), and III (12% [20/172 isolates]). This study shows that Bangladesh has all of the ingredients for invasive GBS disease, including colonization of mothers by invasive serotypes and vertical transmission to babies.

Mechanism of Escherichia coli resistance to Pyrrhocoricin.

Due to their lack of toxicity to mammalian cells and good serum stability, proline-rich antimicrobial peptides (PR-AMPs) have been proposed as promising candidates for the treatment of infections caused by antimicrobial-resistant bacterial pathogens. It has been hypothesized that these peptides act on multiple targets within bacterial cells, and therefore the likelihood of the emergence of resistance was considered to be low. Here, we show that spontaneous Escherichia coli mutants resistant to pyrrhocoricin arise at a frequency of approximately 6 × 10(-7). Multiple independently derived mutants all contained a deletion in a nonessential gene that encodes the putative peptide uptake permease SbmA. Sensitivity could be restored to the mutants by complementation with an intact copy of the sbmA gene. These findings question the viability of the development of insect PR-AMPs as antimicrobials.

Guanidinium Chloride-Induced Haemolysis Assay to Measure New Permeation Pathway Functionality in Rodent Malaria Plasmodium berghei.

Parasite-derived new permeation pathways (NPPs) expressed at the red blood cell (RBC) membrane enable Plasmodium parasites to take up nutrients from the plasma to facilitate their survival. Thus, NPPs represent a potential novel therapeutic target for malaria. The putative channel component of the NPP in the human malaria parasite P. falciparum is encoded by mutually exclusively expressed clag3.1/3.2 genes. Complicating the study of the essentiality of these genes to the NPP is the addition of three clag paralogs whose contribution to the P. falciparum channel is uncertain. Rodent malaria P. berghei contains only two clag genes, and thus studies of P. berghei clag genes could significantly aid in dissecting their overall contribution to NPP activity. Previous methods for determining NPP activity in a rodent model have utilised flux-based assays of radioisotope-labelled substrates or patch clamping. This study aimed to ratify a streamlined haemolysis assay capable of assessing the functionality of P. berghei NPPs. Several isotonic lysis solutions were tested for their ability to preferentially lyse infected RBCs (iRBCs), leaving uninfected RBCs (uRBCs) intact. The osmotic lysis assay was optimised and validated in the presence of NPP inhibitors to demonstrate the uptake of the lysis solution via the NPPs. Guanidinium chloride proved to be the most efficient reagent to use in an osmotic lysis assay to establish NPP functionality. Furthermore, following treatment with guanidinium chloride, ring-stage parasites could develop into trophozoites and schizonts, potentially enabling use of guanidinium chloride for parasite synchronisation. This haemolysis assay will be useful for further investigation of NPPs in P. berghei and could assist in validating its protein constituents.

Impact of introduction of the Haemophilus influenzae type b conjugate vaccine into childhood immunization on meningitis in Bangladeshi infants.

OBJECTIVES: Some Asian countries have been reluctant to adopt Haemophilus influenzae type b (Hib) conjugate vaccination because of uncertainty over disease burden. We assessed the impact of introduction of Hib conjugate vaccine into the Expanded Program on Immunization in Bangladesh on purulent and laboratory-confirmed H influenzae meningitis. STUDY DESIGN: Within a well-defined catchment area around 2 surveillance hospitals in Dhaka, Bangladesh, we compared the incidence of Hib meningitis confirmed by culture, latex agglutination, and polymerase chain reaction assay among infants 1 year before and 1 year after introduction of Hib conjugate vaccine. We adjusted the incidence rate for the proportion of children who sought care at the surveillance hospitals. RESULTS: Among infants, the incidence of confirmed Hib meningitis decreased from 92-16 cases per 100,000 within 1 year of vaccine introduction (vaccine preventable incidence = 76; 95% CI 18, 135 per 100,000). The incidence of purulent meningitis decreased from 1659-1159 per 100,000 (vaccine preventable incidence = 500; 95% CI: 203, 799 per 100,000). During the same time period, there was no significant difference in the incidence of meningitis due to Streptococcus pneumoniae. CONCLUSIONS: Introduction of conjugate Hib conjugate vaccine into Bangladesh Expanded Program on Immunization markedly reduced the burden of Hib and purulent meningitis.

Early-onset neonatal sepsis in Dhaka, Bangladesh: risk associated with maternal bacterial colonisation and chorioamnionitis.

OBJECTIVE: To estimate the risk of early-onset neonatal sepsis among newborns of mothers with chorioamnionitis and/or bacterial colonisation in Dhaka. METHODS: We conducted a cohort study at a maternity centre following 600 mother-newborn pairs. Women with a positive bacterial vaginal culture or positive Group B streptococcus (GBS) rectal culture during labour were classified as colonised. Women with placental histopathology demonstrating signs of maternal or foetal inflammation were classified as having chorioamnionitis. Newborns were followed over the first 7 days of life. The primary outcome measure was physician or community health worker diagnosis of neonatal sepsis following modified World Health Organization Integrated Management of Childhood Illnesses criteria. Survival analysis was conducted with non-parametric, parametric and semiparametric models. RESULTS: Of the 600 mother-newborn pairs, 12.8% of newborns were diagnosed with early-onset sepsis. Five hundred and forty-three women had both colonisation and chorioamnionitis data, 55.4% of mothers were non-exposed, 31.7% were only colonised and 12.9% had chorioamnionitis regardless of colonisation status. After adjusting for birthweight, sex, maternal characteristics and wealth, newborns of only colonised mothers developed sepsis 63% faster and had a 71% higher risk of developing sepsis than their non-exposed counterparts (RT = 0.37, 95% CI 0.14-1.03; RH = 1.71, 95% CI 1.00-2.94). Newborns of mothers with chorioamnionitis developed sepsis 74% faster and had a 111% higher risk of developing sepsis (RT = 0.26, 95% CI 0.07-0.94; RH = 2.11, 95% CI 1.06-4.21). CONCLUSIONS: Newborns born to mothers with colonisation or chorioamnionitis developed sepsis faster and were at higher risk of developing sepsis in Dhaka.

Cloning, purification and preliminary crystallographic analysis of the Helicobacter pylori leucyl aminopeptidase-bestatin complex.

Helicobacter pylori is an important human pathogenic bacterium associated with numerous severe gastroduodenal diseases, including ulcers and gastric cancer. Cytosolic leucyl aminopeptidase (LAP) is an important housekeeping protein that is involved in peptide and protein turnover, catabolism of proteins and modulation of gene expression. LAP is upregulated in metronidazole-resistant H. pylori, which suggests that, in addition to having an important housekeeping role, LAP contributes to the mechanism of drug resistance. Crystals of H. pylori LAP have been grown by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitating agent. The crystals belonged to the primitive triclinic space group P1, with unit-cell parameters a = 97.5, b = 100.2, c = 100.4 Å, α = 75.4, ß = 60.9, γ = 81.8°. An X-ray diffraction data set was collected to 2.8 Šresolution from a single crystal. Molecular-replacement results using these data indicate that H. pylori LAP is a hexamer with 32 symmetry.

Cloning, purification and preliminary crystallographic analysis of the complex of Helicobacter pylori α-carbonic anhydrase with acetazolamide.

Helicobacter pylori infection of the stomach can lead to severe gastroduodenal diseases such as gastritis, peptic ulcers and gastric cancers. Periplasmic H. pylori α-carbonic anhydrase (HpαCA) is essential for the acclimatization of the bacterium to the acidity of the stomach. Through the action of urease and carbonic anhydrases, the H. pylori periplasmic pH is maintained at around 6 in an environment with a pH as low as 2, which in turn facilitates the maintenance of a cytoplasmic pH close to neutral, allowing growth in the gastric niche. Crystals of HpαCA in complex with the inhibitor acetazolamide have been grown by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitating agent. The crystals have the symmetry of space group P2(1)2(1)2(1), with unit-cell parameters a = 37.0, b = 82.4, c = 150.8 Å. An X-ray diffraction data set was collected from a single crystal to 1.7 Šresolution. Calculation of the self-rotation function using this data and molecular replacement showed that the asymmetric unit contains an HpαCA dimer.

Dissecting EXP2 sequence requirements for protein export in malaria parasites.

Apicomplexan parasites that reside within a parasitophorous vacuole harbor a conserved pore-forming protein that enables small-molecule transfer across the parasitophorous vacuole membrane (PVM). In Plasmodium parasites that cause malaria, this nutrient pore is formed by EXP2 which can complement the function of GRA17, an orthologous protein in Toxoplasma gondii. EXP2, however, has an additional function in Plasmodium parasites, serving also as the pore-forming component of the protein export machinery PTEX. To examine how EXP2 can play this additional role, transgenes that encoded truncations of EXP2, GRA17, hybrid GRA17-EXP2, or EXP2 under the transcriptional control of different promoters were expressed in EXP2 knockdown parasites to determine which could complement EXP2 function. This revealed that EXP2 is a unique pore-forming protein, and its protein export role in P. falciparum cannot be complemented by T. gondii GRA17. This was despite the addition of the EXP2 assembly strand and part of the linker helix to GRA17, which are regions necessary for the interaction of EXP2 with the other core PTEX components. This indicates that the body region of EXP2 plays a critical role in PTEX assembly and/or that the absence of other T. gondii GRA proteins in P. falciparum leads to its reduced efficiency of insertion into the PVM and complementation potential. Altering the timing and abundance of EXP2 expression did not affect protein export but affected parasite viability, indicating that the unique transcriptional profile of EXP2 when compared to other PTEX components enables it to serve an additional role in nutrient exchange.

Detection and serotyping of lyophilized nonculturable pneumococcal isolates.

One hundred fifty-two nonculturable lyophilized carriage pneumococcal isolates from a vaccine trial were subjected to PCR for serotyping, and 149 (98%) were successfully classified as vaccine or nonvaccine types, which were similar to viable isolates. The methodology will be useful for analysis of this and other studies where stored pneumococcal isolates fail to grow.

How Malaria Parasites Acquire Nutrients From Their Host.

Plasmodium parasites responsible for the disease malaria reside within erythrocytes. Inside this niche host cell, parasites internalize and digest host hemoglobin to source amino acids required for protein production. However, hemoglobin does not contain isoleucine, an amino acid essential for Plasmodium growth, and the parasite cannot synthesize it de novo. The parasite is also more metabolically active than its host cell, and the rate at which some nutrients are consumed exceeds the rate at which they can be taken up by erythrocyte transporters. To overcome these constraints, Plasmodium parasites increase the permeability of the erythrocyte membrane to isoleucine and other low-molecular-weight solutes it requires for growth by forming new permeation pathways (NPPs). In addition to the erythrocyte membrane, host nutrients also need to cross the encasing parasitophorous vacuole membrane (PVM) and the parasite plasma membrane to access the parasite. This review outlines recent advances that have been made in identifying the molecular constituents of the NPPs, the PVM nutrient channel, and the endocytic apparatus that transports host hemoglobin and identifies key knowledge gaps that remain. Importantly, blocking the ability of Plasmodium to source essential nutrients is lethal to the parasite, and thus, components of these key pathways represent potential antimalaria drug targets.

Antibacterial activity of ethoxzolamide against Helicobacter pylori strains SS1 and 26695.

With the rise of bacterial resistance to conventional antibiotics, re-purposing of Food and Drug Administration (FDA) approved drugs currently used to treat non-bacteria related diseases as new leads for antibacterial drug discovery has become an attractive alternative. Ethoxzolamide (EZA), an FDA-approved diuretic acting as a human carbonic anhydrase inhibitor, is known to kill the gastric pathogenic bacterium Helicobacter pylori in vitro via an, as yet, unknown mechanism. To date, EZA activity and resistance have been investigated for only one H. pylori strain, P12. We have now performed a susceptibility and resistance study with H. pylori strains SS1 and 26695. Mutants resistant to EZA were isolated, characterized and their genomes sequenced. Resistance-conferring mutations were confirmed by backcrossing the mutations into the parent strain. As with P12, resistance to EZA in strains SS1 and 26695 does not develop easily, since the rate of spontaneous resistance acquisition was less than 10-8. Acquisition of resistance was associated with mutations in 3 genes in strain SS1, and in 6 different genes in strain 26695, indicating that EZA targets multiple systems. All resistant isolates had mutations affecting cell wall synthesis and control of gene expression. EZA's potential for treating duodenal ulcers has already been demonstrated. Our findings suggest that EZA may be developed into a novel anti-H. pylori drug.

Structure-Activity Relationship for Sulfonamide Inhibition of Helicobacter pylori α-Carbonic Anhydrase.

α-Carbonic anhydrase of Helicobacter pylori (HpαCA) plays an important role in the acclimation of this oncobacterium to the acidic pH of the stomach. Sulfonamide inhibitors of HpαCA possess anti-H. pylori activity. The crystal structures of complexes of HpαCA with a family of acetazolamide-related sulfonamides have been determined. Analysis of the structures revealed that the mode of sulfonamide binding correlates well with their inhibitory activities. In addition, comparisons with the corresponding inhibitor complexes of human carbonic anhydrase II (HCAII) indicated that HpαCA possesses an additional, alternative binding site for sulfonamides that is not present in HCAII. Furthermore, the hydrophobic pocket in HCAII that stabilizes the apolar moiety of sulfonamide inhibitors is replaced with a more open, hydrophilic pocket in HpαCA. Thus, our analysis identified major structural features can be exploited in the design of selective and more potent inhibitors of HpαCA that may lead to novel antimicrobials.

Structural basis for substrate specificity of Helicobacter pylori M17 aminopeptidase.

The M17 aminopeptidase from the carcinogenic gastric bacterium Helicobacter pylori (HpM17AP) is an important housekeeping enzyme involved in catabolism of endogenous and exogenous peptides. It is implicated in H. pylori defence against the human innate immune response and in the mechanism of metronidazole resistance. Bestatin inhibits HpM17AP and suppresses H. pylori growth. To address the structural basis of catalysis and inhibition of this enzyme, we have established its specificity towards the N-terminal amino acid of peptide substrates and determined the crystal structures of HpM17AP and its complex with bestatin. The position of the D-phenylalanine moiety of the inhibitor with respect to the active-site metal ions, bicarbonate ion and with respect to other M17 aminopeptidases suggested that this residue binds to the S1 subsite of HpM17AP. In contrast to most characterized M17 aminopeptidases, HpM17AP displays preference for L-Arg over L-Leu residues in peptide substrates. Compared to very similar homologues from other bacteria, a distinguishing feature of HpM17AP is a hydrophilic pocket at the end of the S1 subsite that is likely to accommodate the charged head group of the L-Arg residue of the substrate. The pocket is flanked by a sodium ion (not present in M17 aminopeptidases that show preference for L-Leu) and its coordinating water molecules. In addition, the structure suggests that variable loops at the entrance to, and in the middle of, the substrate-binding channel are important determinants of substrate specificity of M17 aminopeptidases.

Prevalence, Serotype Distribution and Mortality Risk Associated With Group B Streptococcus Colonization of Newborns in Rural Bangladesh.

BACKGROUND: Group B Streptococcus (GBS) is a predominant cause of early-onset neonatal sepsis globally; however, the impact of this organism on the health of newborns in South Asia is debated, due in part to a paucity of community-based assessments. We estimated the prevalence and serotypes of GBS colonization of the umbilical cord stump and the association of colonization with mortality in a population-based cohort of newborns in rural Sylhet District, Bangladesh. METHODS: Umbilical cord swabs were collected from 646 newborns up to 3 times within the first week after birth (ages <24 hours, ~3 days, ~6 days) and processed to identify GBS serotypes. RESULTS: GBS was isolated from 6.3% (41/646) of newborns. Sixty-one percent of the GBS colonization occurred in neonates within 24 hours of delivery. Serotypes VII (37.1%, n = 13/36) and Ia (33.3%, n = 12/36) were the most predominant colonizing GBS isolates. Other detected serotypes were Ib (11.1%, n = 4/36), II (11.1%, n = 4/36), V (5.6%, n = 2/36) and VI (2.8%, n = 1/36). Mortality risk among newborns with GBS colonization was 6.6 (95% confidence interval: 2.1-20.4) times higher than for those without GBS. CONCLUSIONS: The overall prevalence of GBS colonization was lower than in settings, where GBS is a predominant etiology of neonatal illness. In addition, the GBS serotype distribution differed from that reported in the developed part of the world. However, further studies are needed to understand the true burden of GBS-related illness. Mortality risk was substantially increased in the presence of GBS on the umbilical stump, providing support for chlorhexidine antisepsis to the cord to prevent colonization of invasive pathogens.

Laboratory Methods for Determining Etiology of Neonatal Infection at Population-based Sites in South Asia: The ANISA Study.

BACKGROUND: The Aetiology of Neonatal Infection in South Asia (ANISA) study aims to determine the etiology of neonatal infections in 5 population-based sites in Bangladesh, India and Pakistan. METHODS: The main laboratory challenges in ANISA were selection and consistent implementation of laboratory methods at participating sites with varied infrastructure. The other specific challenges included (1) specimen collection and transport to designated study laboratories and timely processing in rural settings; (2) minimal or nonexistent laboratory facilities at the field sites; (3) obtaining sufficient volumes of blood from enrolled infants aged 0-59 days and (4) caregivers' concerns about collection of clinical specimens from young infants. An additional challenge was selecting an appropriate molecular platform from multiple available options, all with limited field validation, for use in determining infection in young infants. CONCLUSIONS: This article describes how the challenges of specimen collection, transport and processing and implementation of laboratory methods have been addressed in the ANISA study. It also describes the measures taken to improve detection of microorganisms causing young infant infections by enhancing the sensitivity of existing laboratory methods for pathogen detection.

Structural Basis for the Inhibition of Helicobacter pylori α-Carbonic Anhydrase by Sulfonamides.

Periplasmic α-carbonic anhydrase of Helicobacter pylori (HpαCA), an oncogenic bacterium in the human stomach, is essential for its acclimation to low pH. It catalyses the conversion of carbon dioxide to bicarbonate using Zn(II) as the cofactor. In H. pylori, Neisseria spp., Brucella suis and Streptococcus pneumoniae this enzyme is the target for sulfonamide antibacterial agents. We present structural analysis correlated with inhibition data, on the complexes of HpαCA with two pharmacological inhibitors of human carbonic anhydrases, acetazolamide and methazolamide. This analysis reveals that two sulfonamide oxygen atoms of the inhibitors are positioned proximal to the putative location of the oxygens of the CO2 substrate in the Michaelis complex, whilst the zinc-coordinating sulfonamide nitrogen occupies the position of the catalytic water molecule. The structures are consistent with acetazolamide acting as site-directed, nanomolar inhibitors of the enzyme by mimicking its reaction transition state. Additionally, inhibitor binding provides insights into the channel for substrate entry and product exit. This analysis has implications for the structure-based design of inhibitors of bacterial carbonic anhydrases.

Detection of co-colonization with Streptococcus pneumoniae by algorithmic use of conventional and molecular methods.

Detection of pneumococcal carriage by multiple co-colonizing serotypes is important in assessing the benefits of pneumococcal conjugate vaccine (PCV). Various methods differing in sensitivity, cost and technical complexity have been employed to detect multiple serotypes of pneumococcus in respiratory specimens. We have developed an algorithmic method to detect all known serotypes that preserves the relative abundance of specific serotypes by using Quellung-guided molecular techniques. The method involves culturing respiratory swabs followed by serotyping of 100 colonies by either capsular (10 colonies) or PCR (90 colonies) reactions on 96-well plates. The method was evaluated using 102 nasal swabs from children carrying pneumococcus. Multiple serotypes were detected in 22% of carriers, compared to 3% by World Health Organization (WHO)-recommended morphology-based selection of 1 to 3 colonies. Our method, with a processing cost of $87, could detect subdominant strains making up as low as 1% of the population. The method is affordable, practical, and capable of detecting all known serotypes without false positive reactions or change in the native distribution of multiple serotypes.

Biomarkers for diagnosis of neonatal infections: A systematic analysis of their potential as a point-of-care diagnostics.

BACKGROUND: Neonatal infections annually claim lives of 1.4 million neonates worldwide. Until now, there is no ideal diagnostic test for detecting sepsis and thus management of possible sepsis cases often depends on clinical algorithm leading to empirical treatment. This often results in unnecessary antibiotic use, which may lead to emergence of antibiotic resistance. Biomarkers have shown great promise in diagnosis of sepsis and guiding appropriate treatment of neonates. In this study, we conducted a literature review of existing biomarkers to analyze their status for use as a point-of-care diagnostic in developing countries. METHODS: PubMed and EMBASE database were searched with keywords, 'infections', 'neonates', and 'biomarkers' to retrieve potentially relevant papers from the period 1980 to 2010. Leading hospitals and manufacturers were communicated to inquire about the cost, laboratory requirements and current standing of biomarkers in clinical use. RESULTS: The search returned 6407 papers on biomarkers; 65 were selected after applying inclusion and exclusion criteria. Among the studies, C-reactive protein (CRP), procalcitonin (PCT) and interleukin 6 (IL-6) were the most widely studied biomarkers and were considered to be most promising for diagnosing neonatal infections. About 90% of the studies were from developed countries; more than 50% were from Europe. CONCLUSIONS: Extensive work is being performed to find the diagnostic and prognostic value of biomarkers. However, the methodologies and study design are highly variable. Despite numerous research papers on biomarkers, their use in clinical setting is limited to CRP. The methods for detection of biomarkers are far too advanced to be used at the community level where most of the babies are dying. It is important that a harmonized multi-site study is initiated to find a battery of biomarkers for diagnosis of neonatal infections.