Kidney disease and COVID-19 disease severity-systematic review and meta-analysis.

We aimed to identify prevalence and association of comorbid chronic kidney disease (CKD), acute kidney injury (AKI) and utilization prevalence of continuous renal replacement therapy (CRRT) in COVID-19-hospitalized patients as a function of severity status. With the ongoing struggle across the globe to combat COVID-19 disease, published literature has described the role of kidney disease in COVID-19 patients based on single/multicenter experiences across the globe. We extracted data from observational studies describing comorbid CKD, AKI and CRRT and outcomes and severity of COVID-19-hospitalized patients from December 1, 2019-August 20, 2020 following PRISMA guidelines. Severity of COVID-19 includes intensive care unit admission, oxygen saturation < 90%, invasive mechanical ventilation utilization, in-hospital admission and mortality. Meta-analysis was performed using a random-effects model to calculate pooled estimates, and forest plots were created. In total, 29 studies with 15,017 confirmed COVID-19 patients were included. The overall prevalence of AKI was 11.6% [(430/3693)], comorbid CKD 9.7% [(1342/13,728)] and CRRT 2.58% [(102/3946)] in our meta-analysis. We also found higher odds of comorbid CKD (pooled OR: 1.70; 95%CI: 1.21-2.40; p = 0.002), AKI (8.28; 4.42-15.52; p < 0.00001) and utilization of CRRT (16.90; 9.00-31.74; p < 0.00001) in patients with severe COVID-19 disease. Conclusion Our meta-analysis suggests that comorbid CKD, AKI and utilization of CRRT were significantly associated with COVID-19 disease severity. Clinicians should focus on early triaging of COVID-19 patients with comorbid CKD and at risk for AKI to prevent complication and mortality.

Diagnosis of Helicobacter pylori: what should be the gold standard?

Since the discovery of Helicobacter pylori (H. pylori) in 1983, numerous detection methods for the presence of the bacterium have been developed. Each one of them has been associated with advantages and disadvantages. Noninvasive tests such as serology, (13)C urea breath test (UBT) and stool antigen tests are usually preferred by the clinicians. Serology has its own limitation especially in endemic areas while (13)C UBT is technically very demanding. The stool antigen detection method, although specific, is usually associated with poor sensitivity. The (13)C UBT is believed to be specific, but with present revelation of the fact that stomach is colonized by many other urease producing bacteria makes it questionable. Histology, culture, rapid urease test and polymerase chain reaction (PCR) are the tests which are carried out on antral biopsies collected by invasive means. Histology has been proposed to be very sensitive and specific but the question is how by simply looking the morphology of the bacteria in the microscope, one can claim that the curved bacterium is exclusively H. pylori. Rapid urease test (RUT), the doctor's test, is also challenged because the presence of other urease producing bacteria in the stomach cannot be denied. Moreover, RUT has been reported with poor sensitivity specially, when density of the bacterium is low. Isolation of H. pylori is essential to investigate its growth requirements, antibiotic susceptibility testing, studying virulence factor to develop vaccine and many more explorations. It has also got several disadvantages i.e., special condition for transporting, media, incubation and few days waiting for the colonies to appear, apart from the speed essentially needed to process the specimens. Till date, majority of the microbiological laboratories in the world are not equipped and trained to isolate such fastidious bacterium. The option left is PCR methods to detect H. pylori's DNA in gastric mucosa, gastric juice, saliva, dental plaques and environmental specimens. There are speculations for false positivity due to detection of non-pylori Helicobacters due to genetic sharing; and false negativity due to low bacterial counts and presence of PCR inhibitors. However, specimen collection, transportation and processing do not require speed and special conditions. PCR based diagnosis may be considered as gold standard by designing primers extremely specific to H. pylori and targeting at least more than one conserved genes. Similarly specificity of PCR may be improved by use of internal Primers. Further, nested PCR will take care of false negatives by countering the effect of PCR inhibitors and low bacterial counts. Therefore, nested PCR based methods if performed properly, may be proposed as gold standard test.

Acid exposure induces multiplication of Salmonella enterica serovar Typhi.

Salmonella enterica serovar Typhi faces several environmental stresses while going through the stomach (acidic pH) to the small intestine (basic pH) and intracellularly in macrophages (acidic pH) in humans. The acidic pH followed by alkaline pH in the small intestine might be responsible for expression of certain stress-induced genes, resulting in not only better survival but also induction of multiplication and invasion of the bacterium in the small intestine. Based on this hypothesis, we developed a process wherein we exposed the blood, urine, and stool specimens from 90 acute typhoid fever patients and 36 chronic typhoid carriers to acidic pH to see the effect on isolation rate of S. Typhi. About 5 g of freshly passed unpreserved stool, a centrifuged deposit of 15 ml of urine, and 5 ml of blood clot were subjected to 5 ml of Luria-Bertani (LB) broth (pH 3.5) for 20 min, followed by enrichment in bile broth-selenite F broth. When the combined isolation from all 3 specimens, i.e., blood, urine, and stool, after acid exposure was considered, a total of 77.7% of the acute typhoid patients were observed to be positive for the isolation of the S. Typhi serotype, compared to 8.8% by the conventional method. Similarly, 42% (15/36) of chronic carriers yielded positive for S. Typhi growth after acid exposure, compared to 5.5% (2/36) by the conventional method. It therefore can be concluded that acid shock triggers the multiplication of the bacteria, resulting in better isolation rates from blood clot, stool, and urine specimens.

Helicobacter pylori is not eradicated after triple therapy: a nested PCR based study.

Detection of Helicobacter pylori after triple therapy is usually carried out by either rapid urease test (RUT), urea breath test (UBT), histology, bacterial isolation, and single round PCR or serological tests. In this study, antral biopsy specimens from 25 patients were tested for H. pylori by RUT, culture, histology, and nested PCR in their antral biopsy specimens before and after treatment. Three genes, namely, heat shock protein (hsp60), phosphoglucosamine mutase (ureC), and flagellar export ATP synthase (fliI) of H. pylori were targeted. Of the 25 antral biopsy specimens, the RUT, culture, histology, and nested PCR positivity dropped from 81.8% to 12%, 31% to 0%, 100 to 84%, and 100% to 92%, respectively, before and after therapy. Further, hsp60 specific amplicons from 23 out of 25 patients gave identical restriction pattern, while 6 fliI and 1 ureC specific amplicon produced different restriction pattern. Furthermore, variations in fliI gene sequences in H. pylori after treatment were also confirmed by sequencing and compared in silico. Nested PCR based detection of H. pylori is more sensitive method to detect H. pylori after therapy than culture, RUT, and histology. Further, this study suggests that H. pylori is not eradicated completely after triple therapy.

Mix-infection of S. Typhi and ParaTyphi A in Typhoid Fever and Chronic Typhoid Carriers: A Nested PCR Based Study in North India.

INTRODUCTION: Enteric fever is a systemic disease caused by Salmonella organism such as serotypes Typhi and ParaTyphi A, B, C. Salmonella ParaTyphi A contributes more than 50% of all the enteric fever cases and it has recently been projected as an emerging pathogen. MATERIALS AND METHODS: The present study was aimed to detect Salmonella Typhi and ParaTyphi A in urine, blood and stool specimens collected from cases of enteric fever (110), chronic typhoid carriers (46) and healthy controls (75) to explore the possibility of mixed infection by nested PCR. A new nested PCR primer was designed targeting putative fimbrial protein (stkG) gene which is one of the fimbrial gene families to Salmonella ParaTyphi A and for S. Typhi already reported primers targeting flagellin (fliC) gene. RESULTS: Large volume of urine specimens (15 ml) was found to be the best for detection of Salmonella serotypes. The urine sample was found to have mixed-infection by both the serotypes in 40.9% of the cases but lower in blood (27.3%) and stool (13.6%). CONCLUSION: The present study concludes that occurrence of mixed infection may be quite frequent in typhoid and chronic typhoid carriers' individuals, although the reported recent rise in ParaTyphi A incidence may not be real.

Targeting of putative fimbrial gene for detection of S. Typhi in typhoid fever and chronic typhoid carriers by nested PCR.

INTRODUCTION: It is important to identify Salmonella Typhi infection quickly to treat acute fever patients and to prevent transmission by chronic typhoid carriers; therefore, a very specific and sensitive diagnostic technique is highly desirable, especially in endemic areas. The objective of this study was to develop a PCR protocol targeting the putative fimbrial staA gene of S. Typhi. This is a preferred target gene that is specifically amplified in the S. Typhi serotype compared to the commonly targeted fliC gene which may also be amplified from the non-typhoidal Salmonella Munchen serotype. METHODOLOGY: A new nested PCR primer methodology was designed to target the staA gene, which is a member of the fimbrial gene family specific to Salmonella Typhi only. RESULTS: The primers were found to be very specific as the desired amplicon (377 bp) could be generated exclusively from S. Typhi strains including the reference strain (MTCC 3216) and 78 clinical isolates . Restriction digestion with HinfI confirmed the identity of the amplified DNA fragment in clinical specimens of S. Typhi origin.  Furthermore, these primers were able to detect a minimum of three colony forming units per ml (1fg) in spiked blood samples. The detection sensitivity of the described primers is comparable to that of previously published primers targeting fliC gene sequences. CONCLUSIONS: This study indicates that the primers targeting the putative fimbrial staA gene are very specific to the Typhi serotype and may be a better alternative to fliC targeted amplification based diagnosis.

Pseudomonas fluorescens-like bacteria from the stomach: a microbiological and molecular study.

AIM: To characterize oxidase- and urease-producing bacterial isolates, grown aerobically, that originated from antral biopsies of patients suffering from acid peptic diseases. METHODS: A total of 258 antral biopsy specimens were subjected to isolation of bacteria followed by tests for oxidase and urease production, acid tolerance and aerobic growth. The selected isolates were further characterized by molecular techniques viz. amplifications for 16S rRNA using universal eubacterial and HSP60 gene specific primers. The amplicons were subjected to restriction analysis and partial sequencing. A phylogenetic tree was generated using unweighted pair group method with arithmetic mean (UPGMA) from evolutionary distance computed with bootstrap test of phylogeny. Assessment of acidity tolerance of bacteria isolated from antrum was performed using hydrochloric acid from 10(-7) mol/L to 10(-1) mol/L. RESULTS: Of the 258 antral biopsy specimens collected from patients, 179 (69.4%) were positive for urease production by rapid urease test and 31% (80/258) yielded typical Helicobacter pylori (H. pylori) after 5-7 d of incubation under a microaerophilic environment. A total of 240 (93%) antral biopsies yielded homogeneous semi-translucent and small colonies after overnight incubation. The partial 16S rRNA sequences revealed that the isolates had 99% similarity with Pseudomonas species. A phylogenetic tree on the basis of 16S rRNA sequences denoted that JQ927226 and JQ927227 were likely to be related to Pseudomonas fluorescens (P. fluorescens). On the basis of HSP60 sequences applied to the UPGMA phylogenetic tree, it was observed that isolated strains in an aerobic environment were likely to be P. fluorescens, and HSP60 sequences had more discriminatory potential rather than 16S rRNA sequences. Interestingly, this bacterium was acid tolerant for hours at low pH. Further, a total of 250 (96.9%) genomic DNA samples of 258 biopsy specimens and DNA from 240 bacterial isolates were positive for the 613 bp amplicons by targeting P. fluorescens-specific conserved putative outer membrane protein gene sequences. CONCLUSION: This study indicates that bacterial isolates from antral biopsies grown aerobically were P. fluorescens, and thus acid-tolerant bacteria other than H. pylori can also colonize the stomach and may be implicated in pathogenesis/protection.

Distribution of Hepatitis B virus genotypes among healthy blood donors in eastern part of North India.

AIM: We evaluated the distribution HBV genotypes among non-remunerated healthy blood donors in eastern North India. MATERIALS AND METHODS: During screening of donated blood, 176 consecutive HBsAg positive, samples comprised the study. HBV-DNA was quantitative detected in 150 samples by PCR. HBV genotype was determined by identifying genotype-specific DNA band using nested PCR. RESULTS: Majorities were of age group 31-40 yrs (65.3%). Males (92.7%) outnumbered females (7.3%) and were HbeAg-negative HBsAg carriers. Over all, genotype-A was the most prevalent (54%) followed by D (21.3%). We did not find genotype-G and H. Districts under study, divided into four zones: Zone-I genotype-A was most common (62.3%) followed by D (18.8%); Zone-II genotype-C (41.2%) was more frequent followed by D (20.6% and A (17.7%). Zone-III in adjoining Bihar state close to Zone-I, A was more prevalent (81.8%) followed by B and C (9.1%). In Zone-IV adjoining Zone- II had genotype-A (100%) only. Genotype-D had more sporadic distribution. Genotype-E and F were prevalent in Zone I and II (3/150, 2%). CONCLUSIONS: Among blood donors HBV genotype-A followed by D was the most prevalent in eastern North India. Genotype-A had pattern of distribution signifying common focus, while D was more sporadic and C had single large pocket (Zone-II) probably common focus but restricting to particular area. Evidences are suggestive of association of HBV genotype in liver dysfunction. An effective treatment and preventive strategies based of genotypes will reduce the disease burden and increase the blood safety.