High prevalence of extensively drug resistant and extended spectrum beta lactamases (ESBLs) producing uropathogenic Escherichia coli isolated from Faisalabad, Pakistan.

Urinary tract infections (UTIs) are predominantly caused by uropathogenic Escherichia coli (E. coli). There is rapid increase in antimicrobial resistance in UTIs, also declared as a serious health threat by World Health Organization (WHO). Present study was designed to investigate the antimicrobial resistance status with specific focus on ESBLs and carbapenemases in local uropathogenic E. coli (UPEC) isolates. E. coli isolates were characterized from patients of all ages visiting diagnostic laboratories for urine examination. Demographic data was also recorded for each patient. Antibiograms were developed to observe antibiotic resistance in UPEC using Kirby Bauer disc diffusion technique. Double Disc Synergy test (DDST) was used for phenotypic ESBL test. ESBLs and carbapenemases genes were detected in UPEC using PCR. The PCR results were confirmed by sequencing. The UPEC isolates under study exhibited 78%, 77%, 74%, 72% and 55% resistance against cefotaxime, amoxicillin, erythromycin, ceftriaxone and cefixime, respectively. Resistance against colistin and meropenem was observed in 64% and 34% isolates, respectively. Phenotypic DDST identified 48% isolates as ESBLs producers. Genotypic characterization identified 70%, 74.4% and 49% prevalence of CTXM-1, TEM-1 and CTXM-15 genes respectively. One isolate was observed exhibiting co-existence of all ESBL genes. TEM-1 + CTXM-1 and TEM-1 + CTXM-1 + CTXM-15 + OXA-1 gene patterns were dominant among ESBLs. For carbapenem-resistance, 14% isolates indicated the presence of KPC whereas GES and VIM was detected in 7% and 3.4% isolates, respectively. In conclusion, our results present a high prevalence of extensively drug resistant UPEC isolates with a considerable percentage of ESBL producers. These findings propose the need of continuous surveillance for antimicrobial resistance and targeted antimicrobial therapy.

Biofilm Formation by Staphylococcus epidermidis and Its Inhibition Using Carvacrol, 2-Aminobenzemidazole, and 3-Indole Acetonitrile.

Biofilm-associated bacterial infections are problematic for physicians due to high antimicrobial resistance in biofilm-forming bacteria. Staphylococcus species, particularly Staphylococcus epidermidis, cause severe infections particularly associated with clinical implants. In this study, we have detected the biofilm formation potential of clinical S. epidermidis isolates using phenotypic and genotypic approaches in nutrient-rich and nutrient-deficient growth conditions. The Congo red agar method determined the biofilm formation potential with limited efficacy. However, the tissue culture plate method adroitly classified the isolates as strong, moderate, weak, and non-biofilm producers with five (10%) of the isolates as strong biofilm producers. Ten biofilm-associated genes were targeted, and the fruA gene was found to be the most prevalent (20%). Three antibiofilm compounds, carvacrol, 2-aminobenzemidazole, and 3-indole acetonitrile, were assessed against strong biofilm-producing S. epidermidis isolates. To the best of our knowledge, this is the first report of genotypic and phenotypic detection of biofilms formed by clinical S. epidermidis isolates from this region. The use of 3-indole acetonitrile against these biofilms and toluene as a solvent is novel. The study highlights the significance of biofilm and antibiofilm potential of the studied compounds for effective treatment and control of S. epidermidis infections.

Gene expression and transcriptional regulation driven by transcription factors involved in congenital heart defects.

BACKGROUND: Congenital heart disease (CHD) is one of the most important birth defects caused by more than one mutated gene. Mutations in the genes could cause different types of congenital heart defects including atrial septal defect (ASD), tetralogy of Fallot (TOF), and ventricular septal defect (VSD). OBJECTIVES: Cardiac transcription factors are key players for heart development and are actively involved in controlling stress regulation of the heart. Transcription factors are sequence-specific DNA binding proteins that control the process of transcription and work in a synergistic manner. We aim to characterize core cardiac transcription factors including NKX2-5, TBX, SRF, GATA4, and MEF2, which encode homeobox and MADS domain and play a crucial role in heart development. METHODS: In this study, we have explored the important transcription factors involved in cardiac development and genes controlling the expression and regulation process by using the bioinformatics approach. RESULTS: We have predicted the orthologs and homologs based on their evolutionary history, conserved protein domains, functional sites, and 3D structures for better understanding and presentation of factors responsible for causing CHD. Results showed the importance of these transcription factors for normal heart functioning and development. CONCLUSION: Understanding the molecular pathways and genetic basis of CHD will help to open a new door for the treatment of patients with cardiac defects.

Surface-enhanced Raman spectroscopy for the characterization of pellets of biofilm forming bacterial strains of Staphylococcus epidermidis.

BACKGROUND: Surface-enhanced Raman spectroscopy (SERS) is an effective tool for identifying biofilm forming bacterial strains. Biofilm forming bacteria are considered a major issue in the health sector because they have strong resistance against antibiotics. Staphylococcus epidermidis is commonly present on intravascular devices and prosthetic joints, catheters and wounds. OBJECTIVES: To identify and characterize biofilm forming and non-biofilm forming bacterial strains, surface- enhanced Raman spectroscopy with principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) were used. METHODS: Surface-enhanced Raman spectroscopy (SERS) with silver nanoparticles were employed for the analysis and characterization of biofilm forming bacterial strains. SERS is used to differentiate between non biofilm forming (five samples), medium biofilm forming (five samples) and strong biofilm forming (five samples) bacterial strains by applying silver nanoparticles (AgNPs) as SERS substrate. Principal component analysis (PCA) and Partial least square discriminant analysis (PLS-DA) were used to discriminate between non, medium and strong biofilm ability of bacterial strains. RESULTS: Principal component analysis (PCA) and Partial least square discriminant analysis (PLS-DA) have been used to identify the biochemical differences in the form of SERS features which can be used to differentiate between biofilm forming and non-biofilm forming bacterial strains. PLS-DA provides successful differentiation and classification of these different strains with 94.5% specificity, 96% sensitivity and 89% area under the curve (AUC). CONCLUSIONS: Surface-enhanced Raman spectroscopy can be utilized to differentiate between non, medium and strong biofilm forming bacterial strains.

Vaccinomics to Design a Multiepitope Vaccine against Legionella pneumophila.

Legionella pneumophila is found in the natural aquatic environment and can resist a wide range of environmental conditions. There are around fifty species of Legionella, at least twenty-four of which are directly linked to infections in humans. L. pneumophila is the cause of Legionnaires' disease, a potentially lethal form of pneumonia. By blocking phagosome-lysosome fusion, L. pneumophila lives and proliferates inside macrophages. For this disease, there is presently no authorized multiepitope vaccine available. For the multi-epitope-based vaccine (MEBV), the best antigenic candidates were identified using immunoinformatics and subtractive proteomic techniques. Several immunoinformatics methods were utilized to predict B and T cell epitopes from vaccine candidate proteins. To construct an in silico vaccine, epitopes (07 CTL, 03 HTL, and 07 LBL) were carefully selected and docked with MHC molecules (MHC-I and MHC-II) and human TLR4 molecules. To increase the immunological response, the vaccine was combined with a 50S ribosomal adjuvant. To maximize vaccine protein expression, MEBV was cloned and reverse-translated in Escherichia coli. To prove the MEBV's efficacy, more experimental validation is required. After its development, the resulting vaccine is greatly hoped to aid in the prevention of L. pneumophila infections.

Development of a Candidate Multi-Epitope Subunit Vaccine against Klebsiella aerogenes: Subtractive Proteomics and Immuno-Informatics Approach.

Klebsiella aerogenes is a Gram-negative bacterium which has gained considerable importance in recent years. It is involved in 10% of nosocomial and community-acquired urinary tract infections and 12% of hospital-acquired pneumonia. This organism has an intrinsic ability to produce inducible chromosomal AmpC beta-lactamases, which confer high resistance. The drug resistance in K. aerogenes has been reported in China, Israel, Poland, Italy and the United States, with a high mortality rate (~50%). This study aims to combine immunological approaches with molecular docking approaches for three highly antigenic proteins to design vaccines against K. aerogenes. The synthesis of the B-cell, T-cell (CTL and HTL) and IFN-γ epitopes of the targeted proteins was performed and most conserved epitopes were chosen for future research studies. The vaccine was predicted by connecting the respective epitopes, i.e., B cells, CTL and HTL with KK, AAY and GPGPG linkers and all these were connected with N-terminal adjuvants with EAAAK linker. The humoral response of the constructed vaccine was measured through IFN-γ and B-cell epitopes. Before being used as vaccine candidate, all identified B-cell, HTL and CTL epitopes were tested for antigenicity, allergenicity and toxicity to check the safety profiles of our vaccine. To find out the compatibility of constructed vaccine with receptors, MHC-I, followed by MHC-II and TLR4 receptors, was docked with the vaccine. Lastly, in order to precisely certify the proper expression and integrity of our construct, in silico cloning was carried out. Further studies are needed to confirm the safety features and immunogenicity of the vaccine.

Disease-associated variants of Gap Junction Beta 2 protein (GJB2) in the deaf population of Southern Punjab of Pakistan.

Hearing impairment (HI) is a highly heterogeneous genetic disorder and is classified into nonsyndromic (without any other clinical manifestations) and syndromic (if combined with other clinical presentations) forms. Variations in GJB2 gene are the leading cause of autosomal recessive nonsyndromic hearing loss (ARNSHL) in several populations worldwide. This study was carried out to investigate the prevalence of GJB2 variations in severe-to-profound hearing impaired families of Southern Punjab of Pakistan. Ten families segregating ARNSHL were recruited from different areas of the region. Sanger sequencing of GJB2 coding region was carried out. In two out of ten families, NM_004004:c.*71G>A (p.(Trp24*)) and NM_004004:c.358_360del (p.(Glu120del)) homozygous variants were identified as the cause of hearing loss. Our study showed that GJB2-related hearing loss accounts for at least 20% of all cases with severe-to-profound hearing loss in the Southern Punjab population of Pakistan.

Pathogenic variants of AIPL1, MERTK, GUCY2D, and FOXE3 in Pakistani families with clinically heterogeneous eye diseases.

Significant number out of 2.2 billion vision impairments in the world can be attributed to genetics. The current study is aimed to decipher the genetic basis of Leber congenital Amaurosis (LCA), Anterior Segment dysgenesis (ASD), and Retinitis Pigmentosa (RP), segregating in four large consanguineous Pakistani families. The exome sequencing followed by segregation analysis via Sanger sequencing revealed the LCA phenotypes segregating in families GCUF01 and GCUF04 can be attributed to c.465G>T (p.(Gln155His)) missense and novel c.139_140delinsA p.(Pro47Trhfster38) frameshift variant of AIPL1 and GUCY2D, respectively. The c.1843A>T (p.(Lys615*) truncating allele of MERTK is homozygous in all the affected individuals, presumably suffering with RP, of the GCUF02 family. Meanwhile, co-segregation of the ASD phenotype and the c.289A>G (p.(Ile97Val)) variant of FOXE3 was found in the GCUF06 family. All the identified variants were either absent or present in very low frequencies in the control databases. Our in-silico analyses and 3D molecular modeling support the deleterious impact of these variants on the encoded proteins. Variants identified in MERTK, GUCY2D, and FOXE3 were categorized as "pathogenic" or "likely pathogenic", while the missense variant found in AIPL1 was deemed to have "uncertain significance" based upon the variant pathogenicity guidelines from the American College of Medical Genetics and Genomics (ACMG). This paper highlights the genetic diversity of vision disorders in the Pakistani population and reports the identification of four novel mutations in families who segregate clinically heterogeneous eye diseases. Our results give insight into the genotype-phenotype correlations of AIPL1, FOXE3, MERTK, and GUCY2D variants.

Rapid screening of pyogenic Staphylococcus aureus for confirmation of genus and species, methicillin resistance and virulence factors by using two novel multiplex PCR.

OBJECTIVES: Emergence of methicillin resistant Staphylococcus aureus (MRSA) is a major medical problem of current era. These bacteria are resistant to most drugs and rapid diagnosis can provide a clear guideline to clinicians. They possess specific virulence factors and relevant information can be very useful. We designed this study to develop multiplex PCRs to provide rapid information. METHODS: We studied 60 Staphylococcus aureus isolates and detected methicillin resistance by cefoxitin sensitivity and targeting of mecA gene. After initial studies with uniplex PCRs we optimized two multiplex PCRs with highly reproducible results. The first multiplex PCR was developed to confirm genus, species and methicillin resistance simultaneously, and the second multiplex PCR was for screening of virulence factors. RESULTS: We found 38.33% isolates as methicillin resistant. α -toxin, the major cytotoxic factor, was detected in 40% whereas ß-hemolysin was found in 25% cases. Panton Valentine leucocidin was detected in 8.33% and toxic shock syndrome toxin in5% cases. The results of uniplex and multiplex PCRs were highly compatible. CONCLUSIONS: These two multiplex PCRs when run simultaneously can provide vital information about methicillin resistance and virulence status of the isolate within a few hours as compared to several days needed by routine procedures.

Detection of high levels of resistance to linezolid and vancomycin in Staphylococcus aureus.

Both methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) are rapidly overcoming the current array of drugs. One hundred and fifty isolates from a hospital were studied for resistance towards linezolid and vancomycin. Fifty-four (36.0 %) isolates were MRSA. Both MRSA and MSSA showed high resistance towards linezolid when using the disc diffusion method, with the figures being 48.1 and 29.2 %, respectively. The figures for the E-test were 46.3 and 27.0 %, respectively. The vancomycin resistance was remarkable in MRSA (14.8 %), but relatively low in MSSA (3.1 %). The E-test results were 13.0 and 4.16 %, respectively. The cfr gene was detected in 78 % of linezolid-resistant isolates and the vanA operon was detected in 74 % of vancomycin-resistant isolates. This level of resistance against linezolid and vancomycin is unprecedented. These results are alarming and highlight the threat of non-treatable S. aureus strains.

Relationship of oxidative stress with elevated level of DNA damage and homocysteine in cardiovascular disease patients.

Amounts of DNA damage and homocysteine (Hcy) in heart patients blood may have strong function in the causation of cardiovascular disease (CVD). The main objective of this work was to know experimentally the role of total oxidants (produced by Reactive Oxygen species (ROS), clinical biochemical indices, their oxidized products and total antioxidant status (TAS) among such patients to find the association of homocysteine, total oxidation status (TOS) and oxidative DNA damage with other clinical parameters in sixty positive CVD patients compared with those of 60 normal subjects. As compared to healthy individuals, CVD patients had significantly higher concentrations of homocysteine (p<0.0001), total oxidants stress (TOS) (p<0.0001), serum total lipids (p<0.04), malondialdehyde (MDA) (p<0.001), high density lipoprotein-cholesterol (HDL-C) (p<0.0001), and low density lipoprotein cholesterol (LDL-C) (p<0.01), than those of healthy individuals. Plasma Hcy content, TOS and amount of DNA were positively and significantly associated with cholesterol, triglycerides, systolic blood pressure, urea, and albumin (p values<0.01). TOS, Hcy and oxidative DNA damage were negatively correlated with HDL-c, TAS and proteins. It is suggested that these parameters have pivotal role in diagnostic process of determining severity in CAD patients. Oxidized products of macromolecules in blood of CVD patients impart major functions in causing CVD disease.

Phylogenetic and Comparative Sequence Analysis of Thermostable Alpha Amylases of kingdom Archea, Prokaryotes and Eukaryotes.

Alpha amylase family is generally defined as a group of enzymes that can hydrolyse and transglycosylase α-(1, 4) or α-(1, 6) glycosidic bonds along with the preservation of anomeric configuration. For the comparative analysis of alpha amylase family, nucleotide sequences of seven thermo stable organisms of Kingdom Archea i.e. Pyrococcus furiosus (100-105°C), Kingdom Prokaryotes i.e. Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C), Bacillus amyloliquefaciens (72°C), Bacillus subtilis (70°C) and Bacillus KSM K38 (55°C) and Eukaryotes i.e. Aspergillus oryzae (60°C) were selected from NCBI. Primary structure composition analysis and Conserved sequence analysis were conducted through Bio Edit tools. Results from BioEdit shown only three conserved regions of base pairs and least similarity in MSA of the above mentioned alpha amylases. In Mega 5.1 Phylogeny of thermo stable alpha amylases of Kingdom Archea, Prokaryotes and Eukaryote was handled by Neighbor-Joining (NJ) algorithm. Mega 5.1 phylogenetic results suggested that alpha amylases of thermo stable organisms i.e. Pyrococcus furiosus (100-105°C), Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C) and Bacillus amyloliquefaciens (72°C) are more distantly related as compared to less thermo stable organisms. By keeping in mind the characteristics of most thermo stable alpha amylases novel and improved features can be introduced in less thermo stable alpha amylases so that they become more thermo tolerant and productive for industry.

Preparation, biodistribution, and scintigraphic evaluation of (99m)Tc-clindamycin: an infection imaging agent.

Bacterial infection is found to be the cause of death throughout the world. Nuclear medicine imaging with the help of radiopharmaceuticals has great potential for treating infections. In the present work, clindamycin, a lincosamide antibiotic, was labeled with technetium-99 m (~380 MBq). Clindamycin has been proven to be efficient for treating serious infections caused by bacteria such as Staphylococcus aureus. Quality control, characterization, biodistribution, and scintigraphy of radiolabeled clindamycin were done, and labeling efficiency was determined by ascending paper chromatography. More than 95 % labeling efficiency with technetium-99 m ((99m)Tc) was achieved at pH 6-7 while using 2.5-3 µg SnCl2 · H2O as a reducing agent and 100 µg of ligand at room temperature. The characterization of the compound was performed by using electrophoresis, HPLC and shake flask assay. Electrophoresis indicates the neutral behavior of (99m)Tc-clindamycin. HPLC analysis confirms the single specie of the labeled compound, while shake flask assay confirms high lipophilicity. The biodistribution studies of (99m)Tc-clindamycin were performed Sprague Dawley rats bearing bacterial infection. Scintigraphy and biodistribution studies showed a high uptake of (99m)Tc-clindamycin in the liver, heart, lung, and stomach as well as at S. aureus-infected sites in rabbits.

Study of rust resistance genes in wheat germplasm with DNA markers.

Wheat is a vital dietary component for human health and widely consumed in the world. Wheat rusts are dangerous pathogens and contribute serious threat to its production. In present study, PCR-Based DNA Markers were employed to check the rust resistance genes among 20 wheat genotypes and 22 markers were amplified. NTSYS-pc 2.2 was used to calculate genetic diversity and Nei and Li's coefficients ranged from 0.55 to 0.95. Cluster analysis was obtained using UPGMA (Unweighted Pair Group Method of Arithmetic Average) algorithm. Maximum no. of genes (23) was amplified from TW-760010 genotype whereas minimum no of genes (14) were amplified from TW-76005 genotype. The data gained from present study open up new ways to produce new varieties by breeding rust resistant germplasm to avoid the economic and food loss and varieties with improved characteristics.

Global consensus sequence development and analysis of dengue NS3 conserved domains.

The dengue virus (DENV) genome encodes 10 different genes including the NS3 gene, which has a protease and helicase domain used in virus replication. This domain is a potential target for antiviral agents against dengue. Due to a high mutation rate, DENV is classified into four major serotypes (DENV1-DENV4). This study was designed to perform conservancy analysis of all four serotypes by drawing a consensus sequence for each serotype and then drawing a global consensus sequence to study conserved residues in all four serotypes. A total of 127 NS3 sequences belonging to all four serotypes were retrieved and aligned using multiple alignment feature of CLC Workbench and were subjected to phylogenetic tree construction. Conservancy analysis of NS3 revealed conserved peptides with active site residues that can be important in developing antiviral agents against dengue virus. Among conserved residues, residues G142, Ser144, and G145 (catalytic pocket residues), A219, D220, and D221 (divalent cations binding residues), and His56, Asp79, Ser144, 146 were highly conserved among all the serotypes. Residues from L138 to L149 and from L226 to L245 were also considerably conserved in all serotypes, while lysine141 mutated to serine in serotype 3. A total of 14 peptides from the conserved regions of DENV NS3 protein were identified, which may be helpful to develop peptide inhibitors. The DENV NS3 phylogenetic tree showed the evolutionary relationship among all four serotypes, and all serotypes of dengue were found to have evolved from the dengue 4 serotype. Because of its high variability, DENV has become a global health concern. It is important to study residues that are present in protease, helicase, the catalytic pocket Mg(2+) binding site, and the AAA domain. This study revealed peptides with active site residues that are highly conserved among all four serotypes. These regions of the NS3 sequence may be helpful in developing antiviral agents.

HBV induced HCC: major risk factors from genetic to molecular level.

Hepatocellular carcinoma (HCC) is a deadly and emerging disease leading to death in Asian countries. High hepatitis B virus (HBV) load and chronic hepatitis B (CHB) infection increase the risk of developing HCC. HBV is a DNA virus that can integrate DNA into host genome thereby increase the yield of transactivator protein HBxAg that may deregulate many pathways involving in metabolism of cells. Several monogenic and polygenic risk factors are also involved in HCC development. This review summarizes the mechanism involved in HCC development and discusses some promising therapies to make HCC curative.

Effect of biofilm formation on the excretion of Salmonella enterica serovar Typhi in feces.

OBJECTIVES: We hypothesized that Salmonella enterica serovar Typhi (S. Typhi) with higher biofilm and capsule production capability are more able to survive continuously in typhoid patients/carriers, with subsequent prolonged shedding in feces. METHODS: Bacterial cell release from biofilm (produced in vitro and confirmed by specific staining and electron microscopy) and comparative cytotoxicity were studied on Caco2 cells. Functionality of the biofilm diffusion barrier was tested against ciprofloxacin. Biofilm production was graded and semi-quantified as -, +, ++, +++, and ++++. RESULTS: Out of 30 isolates, 23 produced biofilm. The average post-treatment detection of S. Typhi in blood was 7-13 days and in stool was 13-32 days. A fall in cell count from 104 to approximately 10¹ over the course of 3 days as compared to total elimination of planktonic cells in 16 h after ciprofloxacin application substantiated the protective role of biofilm. Lactic dehydrogenase release ranged from 38% in non-biofilm producers to 97% in the highest biofilm producers, indicating increased pathogenic behavior. CONCLUSIONS: The period of S. Typhi clearance from typhoid patients after recovery was found to be directly related to biofilm production capability.

Multiplex PCR for differential diagnosis of emerging typhoidal pathogens directly from blood samples.

Classically Salmonella enterica serovar Typhi (S. Typhi) is associated with typhoid, a major health problem in developing countries. However, in recent years S. Paratyphi A and Vi-negative variants of S. Typhi have emerged rapidly. We have developed a nested multiplex PCR targeting five different genes for differential diagnosis of typhoidal pathogens which has been optimized to be directly applicable on clinical blood samples. Of 42 multiplex PCR-positive blood samples, 26, nine, and two were Vi-positive S. Typhi, Vi-negative S. Typhi and S. Paratyphi A, respectively, and five patients were found to have mixed infection. Seventeen patients grew Salmonella from blood culture and the remaining 25 were positive in the Salmonella-specific PCR. Tests with several common pathogens confirmed the specificity of the assay. We conclude that the proposed multiplex PCR is rapid, sensitive and specific for the diagnosis of typhoidal pathogens directly from blood samples.

Detection of Vi-negative Salmonella enterica serovar typhi in the peripheral blood of patients with typhoid fever in the Faisalabad region of Pakistan.

The synthesis and transportation proteins of the Vi capsular polysaccharide of Salmonella enterica serovar Typhi (serovar Typhi) are encoded by the viaB operon, which resides on a 134-kb pathogenicity island known as SPI-7. In recent years, Vi-negative strains of serovar Typhi have been reported in regions where typhoid fever is endemic. However, because Vi negativity can arise during in vitro passage, the clinical significance of Vi-negative serovar Typhi is not clear. To investigate the loss of Vi expression at the genetic level, 60 stored strains of serovar Typhi from the Faisalabad region of Pakistan were analyzed by PCR for the presence of SPI-7 and two genes essential for Vi production: tviA and tviB. Nine of the sixty strains analyzed (15%) tested negative for both tviA and tviB; only two of these strains lacked SPI-7. In order to investigate whether this phenomenon occurred in vivo, blood samples from patients with the clinical symptoms of typhoid fever were also investigated. Of 48 blood samples tested, 42 tested positive by fliC PCR for serovar Typhi; 4 of these were negative for tviA and tviB. Three of these samples tested positive for SPI-7. These results demonstrate that viaB-negative, SPI-7-positive serovar Typhi is naturally occurring and can be detected by PCR in the peripheral blood of typhoid patients in this region. The method described here can be used to monitor the incidence of Vi-negative serovar Typhi in regions where the Vi vaccine is used.