**4. Use of molecular techniques in chlamydia diagnosis and screening**

Since the late 1990s, chlamydia has been the most commonly reported sexually transmitted infection (STI) in Europe and the United States. The infection is caused by the bacterium *C. trachomatis* (*C. trachomatis*), and its common name was given in the late nineteenth century after an infection-causing pathogen. In 2017, there were just over 203,116 new diagnoses in the UK. In contrast, 7137 syphilis and 44,676 gonorrhea and more than 3,126,000 chlamydia diagnoses were made by the National Chlamydia screening program. The disease was equally prevalent in men, and although similar effects on the seminal vesicles are expected, there is as yet no evidence of a strong association between infection and male infertility. The program began in 2004, and the reported incidence of chlamydia has skyrocketed, with journalists warning of "a time bomb for fertility" from these reports. Chlamydia was first recognized as a specific sexually transmitted infection in the 1970s, but in 1988, it became reportable.

There are different types of screening options for chlamydial infections. It has the highest rate of spread on the scale of infectious diseases among youth and late adults. Chlamydia is easily transmitted sexually and can also be transmitted to newborns. A large number of infectious individuals may be overlooked in screening because they do not have symptoms. Although chlamydia can cause serious infections, it can be easily diagnosed with various new tests with high sensitivity. The new urine tests are rapid and noninvasive and give rapid results. It has been shown that scans reduce the rates of pelvic inflammatory disease (PID) and improve birth outcomes when pregnant women are screened and treated. Cost studies, on the other hand, show that screening for women is more cost-effective than no screening. The evidence for screening in men is quite limited. Little is known about how often any of these groups will be screened. Worse health outcomes, such as recurrent infections, PID in women, and ectopic pregnancies, should be followed by conventions, which are currently largely in the domain of public health programs, not clinical practice. As the responsibility for these tasks shifts to public health organizations, clinicians may become more involved

in these secondary preventive measures [23]. Increasing evidence suggests that the rate of progression of endocervical chlamydia to pelvic inflammatory disease is lower than previously thought. Population-based studies consistently estimate the incidence rates of pelvic inflammatory disease to be lower than that by clinical-based studies. Therefore, infections detected by screening asymptomatic individuals may have a better prognosis than that of symptomatic infections due to differences in the burden of the organism. However, descriptions of chlamydial infection and its consequences and models of the impact of screening almost always refer to higher estimates [14].

Compared to nucleic acid amplification tests, the sensitivity of conventional methods is very low [19].

Commercial polymerase chain reaction (PCR) uses multiple amplifier product line-based methods to target DNA. This is a cryptic plasmid DNA of 207 nucleotides, which is highly conserved among serotypes for *C. trachomatis.*

Chlamydia species are the leading cause of bacterial STDs, important respiratory pathogens, and the etiologic agent of endemic blinding trachoma, a zoonotic threat. In the last decade, molecular genetic analyses of Chlamydia species have advanced rapidly [24]. *C. trachomatis* (Ct) is the most common sexually transmitted disease worldwide. A Ct infection can cause urethritis, cervicitis, proctitis, and conjunctivitis depending on the anatomical site of the infection. It is asymptomatic in 50% of men and 70% of women. When *C. trachomatis* infection is not treated, it can cause infertility in men and women, such as epididymitis and pelvic inflammatory disease [3].

In the study using DNA enzyme immunoassay (DEAI) and reverse hybridization assay techniques, 19 serovar typing was performed by distinguishing it from other bacteria and chlamydia species or commensal microorganisms in the genital tract with very high sensitivity as a result of the identification of *C. trachomatis*. First, PCR products were hybridized with the probe mixes for the cryptic, plasmid, and omp 1 genes, and *C. trachomatis* was detected [18]. *C. trachomatis* is the most common sexually transmitted bacterial infection in the United States, usually by asymptomatic individuals. FDA-approved molecular methods for diagnosing urogenital *C. trachomatis* include nucleic acid hybridization, signal amplification, polymerase chain reaction, chain displacement amplification, and transcription-mediated amplification [25].

Molecular methods are both rapid and reliable for screening genital species in areas with high disease prevalence. The clinical and analytical sensitivity of some tests is seriously reduced when testing from urine samples. In vitro experiments have shown that transcriptional origin amplification is more sensitive than other molecular-based assays [19].

In the detection of *C. trachomatis*, screening tests without amplification include the direct fluorescent antibody test (DFA), optical immunostaining (OIA), and rapid solid-phase enzyme immunoassay (EIA). The accuracy in the diagnosis of *C. trachomatis* using nonmolecular methods confirms the quality of the reference tests [26].

Commercial nucleic acid hybridization is nucleic acid hybridization using oligonucleotide sequences designed to bind to the complementary sequence in the target nucleic acid. It can be used in conjunction with cell culture methods that do not provide optimal conditions due to low sensitivity. In commercial signal amplification, neither target nor oligonucleotide probe nucleic acid concentrations change. It contains targetspecific chromosomal and cryptic plasmid sequences for detecting *C. trachomatis* [19].

*C. trachomatis* infections can be detected using cell culture, immunofluorescence (IF), enzyme immunoassay, direct DNA hybridization, and PCR (identifiable). Laboratory diagnosis of chlamydial infection by culture is limited by the fact that the collection of urethral swab specimens is unacceptable for many asymptomatic men.

PCR applications using various gene targets, such as cryptic plasmid, omp 1 (the gene encoding major outer membrane protein-MOMP), and rRNA genes, are more sensitive than EIR, IA, and culture [10].

*C. trachomatis* is the most common sexually transmitted bacterial infection in the United States, usually by asymptomatic individuals. FDA-approved molecular methods for diagnosing urogenital *C. trachomatis* include nucleic acid hybridization, signal amplification, polymerase chain reaction, chain displacement amplification, and transcription-mediated amplification. Molecular methods are both rapid and reliable for screening genital species in areas with high disease prevalence [27]. The clinical and analytical sensitivity of some tests is seriously reduced when testing from urine samples. In vitro experiments have shown that transcriptional origin amplification is more sensitive than other molecular-based assays [19].

Commercial nucleic acid hybridization is nucleic acid hybridization using oligonucleotide sequences designed to bind to the complementary sequence in the target nucleic acid. It can be used in conjunction with cell culture methods that do not provide optimal conditions due to low sensitivity [19]. Samples isolated from 40 patients with *C. trachomatis* infection were transferred to the laboratory, and the methods obtained from the culture method with chlamydial nucleic acids were compared. By PCR, either the characteristic 7.5 kb plasmid DNA or the 16 Cyclic r RNA gene segment was used for identification. All PCR results were validated in Sothern or dot blot format. As a result, 5 *C. trachomatis* were isolated from 6 samples that were PCR positive. More samples (9) were found to be positive, as nucleic acid sequencing showed rRNA-PCR-amplified products in variants. These data showed that *C. trachomatis* infections in patients were either unrecognized or detected variants carrying the *C. trachomatis* plasmid [28].

Epidemiological studies on molecular typing have been conducted since ancient times. Molecular typing and serotyping of 150 *C. trachomatous* specimens isolated from genital sources of 10 different serovars were compared. The most common omp 1 genotypes, E (51.7%), F (17.3%), D (8.8%), and G (8.4%), were determined from the samples collected over 29 months. Molecular biology methods that require molecular biology techniques and equipment allow typing as well as immunology techniques [29].

A different study used rapid antigen identification to identify *C. trachomatis* infections and compared it with direct fluorescent antibody staining and tissue culture. In a study conducted on 507 patients, the sensitivity was found to be 75%, whereas the specificity was 99% [30].

Chlamydia detection and screening methods made from urine samples are commonly used. Sample supply is easier as noninvasive methods are used for sample collection.

However, PCR can also be used because urine specimens are more convenient to collect and more acceptable to patients. Studies have shown that the application of restriction fragment length polymorphism (RFLP), which is another technique in addition to PCR, facilitates the identification of many serovars that are difficult to identify using PCR [31].

PCR is more sensitive and accurate than other methods in chlamydia samples isolated from endocervical swabs. Cell culture PCR results obtained from urine samples as well as the samples collected from the cervix were compared, and the sensitivity was 87% in culture, 92% in cervix PCR, and 95% in urine PCR. Culture from endocervical and urethral swabs is the gold standard for diagnosing chlamydia. However, its sensitivity is affected by many factors, such as conditions during transportation. Recently, PCR amplification methods and ligase chain reactions have been shown to be more sensitive. In recent years, PCR scans from the urine cervix have offered great advantages. Urine

PCR culture is more sensitive than specific culture. Urine chlamydia screenings are more acceptable in large populations and for asymptomatic detection [32].

*C. trachomatis* (Ct) is an atypical agent for developing acute, subclinical, and chronic conjunctivitis. The chain reaction (PCR) procedure was used for the conjunctivitis test and enzyme-linked fluorescence assay (IFA and ELFA) and molecular analysis of Ct DNA search (Ct DNA) with polymerase among a total of 3520 patients who visited the examination room of the G. d'Annunzio University Eye Clinic between 2006 and 2008 in the study formed with the records of 171 patients with occasional mild, moderate, or severe illness from Chieti, Italy, in a prospective open three-arm study using conventional assays such as immunofluorescence in order to evaluate the presence of *C. trachomatis* against trachoma.

Molecular tests such as the GeneXpert CT/NG test are highly sensitive. However, cost constraints prevent these technologies from being implemented in environments with limited resources. Pooled testing is a strategy to reduce the cost per sample, but the extent of the savings depends on the prevalence of the disease. One study used a pooling strategy based on the identification of sociodemographic and laboratory factors associated with the prevalence of CT/NG in a high-risk area of Zambian female sex workers. Factors associated with positive testing for CT/NG through single mothers' logistic regression modeling conducted from 2016 to 2019 included city, young age, low education, and long-acting reversible contraception. Based on these factors, the study population was divided into high-, medium-, and low-prevalence subgroups. *Trichomonas vaginalis* infection was tested in pools of 3 or 4, respectively, according to bacterial vaginosis and syphilis infection. The fee was reduced from \$18 to \$9.43 per sample in the low-prevalence subgroup. The described checklist tool and pooling approach can be used in a variety of ways. This is especially valuable in areas where resources are limited. It is also important in treating asymptomatic CT/NG infections missed by traditional syndromic management.

The most attractive DNA amplification methods can be recommended for screening trachomatis infections because of their excellent sensitivity and good performance. This scan has been shown before in the FVU (first-void urine) samples. PCR testing with FVU is cost-effective in a low-prevalence population. A side risk factor for *C. trachomatis* infection is infection exceeding 3.9%. Recent research has shown that neoplasia is a risk factor for pregnancy outcomes other than ectopic pregnancy and possibly for cervical development. The cost-effectiveness of screening the sexually active population with DNA amplification methods can be reassessed.

Pathogens were compared using multiplex real-time polymerase chain reaction (PCR) in men with acute urethritis. Test results were compared in 83 patients using urethral swab samples, multiple real-time PCR, and A.F. Genital System tests in men diagnosed with acute urethritis. The pathogen of urethritis was detected in 69 patients with PCR and in 15 patients with AF. Compared with AF genital tract multiplex PCR, its sensitivity is low in male patients with previous acute urethritis [33]. Urethritis in men is one of the most common sexually transmitted diseases, and although there have been important developments in treatment and diagnosis in recent years, the most common method is the use of polymerase chain reactions [34].

Highly conserved Chlamydial proteins can be used as specific markers in the diagnosis of chlamydial and constitute new targets of drugs specific to these bacteria. In total, 59 Chlamydia proteins, 79 Chlamydiaceae proteins, 20 both Chlamydia and Chlamydophila, and 445 ORFs were found to be specific to Protochlamydia [18].

Studies have been conducted using tandem mass spectrometry and affinity chromatography methods for Chlamydia infections, which do not have an effective vaccine yet and have resulted in significant deaths worldwide [35].

*Molecular Approaches to the Diagnosis of* Chlamydia *DOI: http://dx.doi.org/10.5772/intechopen.109746*


**6. Conclusions**

The identification of PYL proteins as ABA receptors has placed a crucial piece of the puzzle into the previous knowledge of the core ABA signalling pathway. As such, the early efforts in the discovery of PYL proteins and the elucidation of their structural mechanisms have been recognised as one of the breakthroughs of the year 2009 by *Science* and *Science Signaling* journals [75, 76]. Subsequently, further structural studies have rapidly emerged to provide a compre‐ hensive understanding of the molecular mechanisms of the entire core ABA signalling pathway, which has led to preliminary developments of novel approaches targeting at ABA signalling for the agricultural enhancement of abiotic stress tolerance. Using several lines of approach, these studies have demonstrated the ability to target ABA signalling for improving stress tolerance. While several studies have shown promising data at the preliminary stage, further testing in crop plants will be necessary to evaluate their agricultural feasibility. Having progressed rapidly since the initial discovery of the PYL proteins, further breakthrough advances in the agricultural improvement of abiotic stress tolerance seems highly conceivable and is much awaited for.


**2.4 Bioimpedance and body composition monitoring**

Bioimpedance monitors can provide quick, cheap, non-invasive bedside measurements of fluid status with good reproducibility. However, until recently the use of bioimpedance has been restricted to a relatively small number of centres with both a clinical and an academic interest in the technology.

In 'single frequency' bioimpedance monitors, a tiny 50 kHz alternating current is passed between a pair of electrodes, usually placed on the hand and foot. Sensing electrodes, placed just inside the current carrying electrodes, measure 'resistance' and 'reactance' to the passage of the current. Resistance and reactance combine to give the overall 'impedance'. An increase in body water makes it easier for current to pass through the body so that resistance decreases. Reactance, which is due to the capacitance of cell membranes, decreases as the number and/or integrity of the membranes decreases.

Single frequency bioimpedance monitors are widely used in health clubs as they can give an estimate of body fat and muscle mass. The technology is also built into bathroom scales where the current is passed between the feet. In dialysis patients, the equations used to derive body composition from the impedance at 50 kHz are unreliable if the patient has an abnormal fluid status.

Prof Antonio Piccoli and co-workers recognised this and developed bioimpedance vector analysis (BIVA) which simply looks at the hydration of the body tissues between electrodes placed on the hand and foot. The resistance and reactance measurements are normalised to height and interpreted using gender-specific nomograms derived from large studies of normal subjects and of populations with altered body composition (Piccoli 1994, 1995). As shown in figure 2, fluid overload is associated with movement of the vector downward and to the left whilst dehydration moves the vector up and to the right.

Bioimpedance monitors can provide quick, cheap, non-invasive bedside measurements of fluid status with good reproducibility. However, until recently the use of bioimpedance has been restricted to a relatively small number of centres with both a clinical and an academic

In 'single frequency' bioimpedance monitors, a tiny 50 kHz alternating current is passed between a pair of electrodes, usually placed on the hand and foot. Sensing electrodes, placed just inside the current carrying electrodes, measure 'resistance' and 'reactance' to the passage of the current. Resistance and reactance combine to give the overall 'impedance'. An increase in body water makes it easier for current to pass through the body so that resistance decreases. Reactance, which is due to the capacitance of cell membranes, decreases as the

Single frequency bioimpedance monitors are widely used in health clubs as they can give an estimate of body fat and muscle mass. The technology is also built into bathroom scales where the current is passed between the feet. In dialysis patients, the equations used to derive body composition from the impedance at 50 kHz are unreliable if the patient has an

Prof Antonio Piccoli and co-workers recognised this and developed bioimpedance vector analysis (BIVA) which simply looks at the hydration of the body tissues between electrodes placed on the hand and foot. The resistance and reactance measurements are normalised to height and interpreted using gender-specific nomograms derived from large studies of normal subjects and of populations with altered body composition (Piccoli 1994, 1995). As shown in figure 2, fluid overload is associated with movement of the vector downward and

Fig. 1. Variation in blood volume with extracellular volume

**2.4 Bioimpedance and body composition monitoring**

number and/or integrity of the membranes decreases.

to the left whilst dehydration moves the vector up and to the right.

interest in the technology.

abnormal fluid status.

Fig. 2. Nomogram used for interpreting single frequency bioimpedance measurements. The outer ellipse encloses 95% of readings for normal subjects.

BIVA could provide practical information on changes in fluid status using very simple equipment that would cost little more than a set of bathroom scales if the market was larger. The reason BIVA cannot give an accurate indication of the patient's normally hydrated weight is clear when you examine Figure 2. The vector shown could be obtained from a slightly overweight subject with normal hydration, from a muscular subject with fluid overload or from an obese subject who is dehydrated. The confusion arises because adipose tissue contains very little intracellular water as fat cells are filled with triglycerides, but does have water in the extracellular space. So, like overhydration, an increase in body fat leads to an increase in the proportion of fluid in the extracellular space leading to a shorter vector.

In whole body bioimpedance spectroscopy (BIS), the electrodes are placed as for BIVA and resistance and reactance is measured over a range of frequencies. The results, together with the height, weight and gender of the subject, are used to compute the intracellular and extracellular water volumes (ECW and ICW). Fluid overload is associated with an increase in the proportion of water in the extracellular space but until recently, it was necessary for the user to decide what this proportion should be at normal hydration. There are a number of published methods for doing this (for example Lindley et al, 2005; Lopot et al, 2002) but they involve comparing dialysis patients with normal controls. As with BIVA, this makes it difficult to assess fluid status in patients with abnormal body composition.

Body composition monitoring (BCM) is the most recent commercially available development in bioimpedance monitoring. It uses the same electronic measurements as BIS to determine ECW and ICW but incorporates additional modelling (Moissl et al, 2006; Chamney et al, 2007) to take account of the amount of body fat the patient actually has,

Management of Fluid Status in

Haemodialysis Patients: The Roles of Technology and Dietary Advice 191

systematically decreasing their target weight could compromise their residual renal

When the BCM measurement is combined with the clinical indicators in Table 1, and knowledge of the patient's usual IDFG, it is possible to customise the target weight as described in 2.1. Introducing BCM has been shown to improve blood pressure control and reduce intradialytic adverse events (Machek, 2010). Ideally BCM should be carried out at least quarterly, though more frequent measurements will be needed for patients who are unwell (especially if admitted to hospital) or who are trying to gain or lose weight. As well as ensuring timely adjustments to the prescribed target weight, BCM gives valuable information on changes in body fat and lean tissue and provides an accurate urea

The patient's hydration status during the interdialytic period depends on both the weight achieved after dialysis and the fluid gained by the patient before the next session. Very high IDFG can make it impossible for the patient to remain close to normal hydration and to control pre-dialysis blood pressure. Another problem, particularly in elderly and malnourished patients, is the inability to tolerate the ultrafiltration rates required to remove a moderate volume of accumulated fluid. Whether the patient is gaining excessive volumes or failing to transfer fluid from the tissues into the circulation sufficiently rapidly, the

A typical haemodialysis patient in the UK accumulates about 2 litres of excess fluid in the intervals between sessions. When they attend for dialysis the machine is programmed to remove the excess fluid by ultrafiltration. Every litre removed in this way will carry with it about 137 mmol of sodium ions, though the actual amount will depend on the serum sodium level at the time the fluid was removed. As 137 mmol is the amount of sodium in 8 g of salt, the typical UK patient loses sodium equivalent to about 16 g of salt at each dialysis session. The body does have 'non-osmotic' sodium stores in tissues such as the skin and connective tissues (Titze, 2008) and it is possible that sodium can be recruited into or removed from these stores to buffer short term fluctuations in serum sodium. However, if the patient is assumed to be in steady state on the timescale of the interdialytic period, they must be making up for the sodium lost by taking in the equivalent of 16 g of salt between sessions. Sodium does come in other forms other than salt, such as sodium bicarbonate, but

If retained in the body, the salt taken in will cause 'osmometric' thirst. Osmometric thirst is triggered when increased osmolarity of the extracellular fluid causes osmoreceptor cells in the hypothalamus to shrink. Volumetric thirst, which occurs when the body loses both water and salt, is triggered when baroreceptors in the atria sense low cardiac return volume. Haemodialysis patients may experience volumetric thirst immediately after dialysis if they are dehydrated, but at other times their thirst is primarily osmometric. Fluid drunk in response to post-dialysis dehydration does not usually lead to increased IDFG as it simply delays osmometric thirst until the patient has consumed enough salt. An important exception to this occurs if a patient with good residual renal function is dehydrated, as they

will need to take in enough fluid to normalise their hydration before diuresis starts.

Osmometric thirst is part of the body's system for maintaining electrolyte balance. If our typical haemodialysis patient is anuric (unable to lose sodium via the kidneys), they will need to take in about one litre of water to dilute every 8g salt consumed to a normal

function, cause cramps and leave them feeling exhausted for hours after dialysis.

distribution volume for use in on-line measurements of dialysis adequacy.

**3.1 Salt and fluid balance: Osmometric thirst and the sodium 'set-point'**

**3. Control of interdialytic fluid gain (IDFG)**

solution is to try and reduce their IDFG.

it is usually combined with chloride.

rather than assuming they have the average amount for a person of their age and gender. Essentially, the BCM model assumes that the body is composed of normally hydrated lean tissue, normally hydrated adipose tissue and excess fluid (or missing fluid if the patient is dehydrated). For any combination of ECW, ICW and weight, there is only one matching combination of lean tissue, fat and excess/missing fluid.

With BCM, we can get an estimate of normal hydration and, for the first time, select a target weight that minimises the unwanted effects of dehydration as well as those of overhydration. Just as the introduction of BVM revealed a cohort of chronically overloaded patients, introducing BCM as part of the assessment of fluid status identified a group who were excessively dehydrated. In some of the cases identified in this way, an increase in target weight led to a reduction in interdialytic fluid gain as the patient's kidneys were able to produce more urine.

Fig. 3. Pre-dialysis systolic blood pressure vs hydration status

Users of BCM soon become aware that blood pressure can be misleading when evaluating fluid status. The scatter plot in Figure 3 shows the relationship between pre-dialysis systolic blood pressure and hydration status (the difference between measured and normal hydration in litres), for the first BCM measurement made in 474 haemodialysis patients under the care of Leeds Teaching Hospitals. A very similar plot was obtained in a crosssectional study of 639 PD patients (Van Biesen et al, 2010). The expected increase in blood pressure with fluid overload is present, but only as a trend for the population. For an individual patient, a single high or low blood pressure measurement is a rather poor predictor of fluid status, though a trend to higher or lower blood pressures in the same patient does provide important clinical information.

A significant number of patients who are normally hydrated, or even dehydrated, predialysis have high blood pressure. The traditional method for treating these patients by

rather than assuming they have the average amount for a person of their age and gender. Essentially, the BCM model assumes that the body is composed of normally hydrated lean tissue, normally hydrated adipose tissue and excess fluid (or missing fluid if the patient is dehydrated). For any combination of ECW, ICW and weight, there is only one matching

With BCM, we can get an estimate of normal hydration and, for the first time, select a target weight that minimises the unwanted effects of dehydration as well as those of overhydration. Just as the introduction of BVM revealed a cohort of chronically overloaded patients, introducing BCM as part of the assessment of fluid status identified a group who were excessively dehydrated. In some of the cases identified in this way, an increase in target weight led to a reduction in interdialytic fluid gain as the patient's kidneys were able

combination of lean tissue, fat and excess/missing fluid.

Fig. 3. Pre-dialysis systolic blood pressure vs hydration status

patient does provide important clinical information.

Users of BCM soon become aware that blood pressure can be misleading when evaluating fluid status. The scatter plot in Figure 3 shows the relationship between pre-dialysis systolic blood pressure and hydration status (the difference between measured and normal hydration in litres), for the first BCM measurement made in 474 haemodialysis patients under the care of Leeds Teaching Hospitals. A very similar plot was obtained in a crosssectional study of 639 PD patients (Van Biesen et al, 2010). The expected increase in blood pressure with fluid overload is present, but only as a trend for the population. For an individual patient, a single high or low blood pressure measurement is a rather poor predictor of fluid status, though a trend to higher or lower blood pressures in the same

A significant number of patients who are normally hydrated, or even dehydrated, predialysis have high blood pressure. The traditional method for treating these patients by

to produce more urine.

systematically decreasing their target weight could compromise their residual renal function, cause cramps and leave them feeling exhausted for hours after dialysis.

When the BCM measurement is combined with the clinical indicators in Table 1, and knowledge of the patient's usual IDFG, it is possible to customise the target weight as described in 2.1. Introducing BCM has been shown to improve blood pressure control and reduce intradialytic adverse events (Machek, 2010). Ideally BCM should be carried out at least quarterly, though more frequent measurements will be needed for patients who are unwell (especially if admitted to hospital) or who are trying to gain or lose weight. As well as ensuring timely adjustments to the prescribed target weight, BCM gives valuable information on changes in body fat and lean tissue and provides an accurate urea distribution volume for use in on-line measurements of dialysis adequacy.


**Author details**

Tulshi Shiyani Saurashtra Research Institute, SRI, Rajkot, India

\*Address all correspondence to<EMAIL_ADDRESS>
© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


**2. Experimental Procedure**

The 6mm thick AA1050-H14 and AA5083-H111 plates were cut into 70 mm × 530 mm long strips using a guillotine cutting machine (see Figure 1).

**Figure 1.** Aluminium alloy plates prepared for welding.

The two dissimilar plates were fixed on the backplate of the semi-automated milling machine (FSW machine), as shown in Figure 2. The AA5083-H111 was kept on the retreating side throughout the experiments, while AA1050-H14 was on the advancing side. This kind of material location was followed based on the recommendation from the literature [11,12,16,19,25,26]. The plates were fixed on the machine's backplate by eight clamps. The function of clamps was to make sure the plates did not move apart when the rotating pin was in motion.

**Figure 2.** Friction stir welding machine.

The plates were then friction stir welded using a high carbon steel (H13) tool shown in Figure 3. The tool was machined using a lathe machine and was heat-treated to about 50HRC. The profile of the pin was triangularly threaded with a 20 mm shoulder diameter and 6 mm pin diameter. The triangular threaded pin had a 1mm pitch and the height of 5.8 mm. The FSW parameters used in this study are presented in Table 1, and these parameters were chosen based on various speed test combinations performed prior to the main welding. However, those trial results are not included in this work since they are not part of the paper's focus.

**Figure 3.** Friction stir welding tool.

Figure 4 shows the friction stir welding of 6 mm thick AA1050 and AA5083 plates. Stage 1 shows two plates ready for welding. Stage 2 shows the rotating tool plunged into the two pieces being welded, i.e., AA1050-H14 and AA5083-H111. Stage 3 shows the welded part of the plates. Stage 4 shows the end of FSW and the unplunging of the tool. The finished product is clearly shown in Figure 5. The nominal chemical composition for materials used in this study is presented in Table 2. The chemical composition was measured using the BELEC COMPACT PORT HLC spectrometer (Belec Spektrometrie Opto-Elektronik GmbH, Georgsmarrienhutte, Germany) according to ASTM E716-16 standard. These chemical compositions were in the range of those reported in the literature [16,21,27].

**Figure 4.** Friction stir welding process.

**Figure 5.** FSW joint.


**2. Developmental aspects of meninges pertinent to primary ectopic meningiomas and ectopic meningothelial tissue**

This will be a brief section and by no means an exhaustive treatment of the embryological, morphological and molecular genetics aspects of meninges development. For a more thorough review of meninges development, the reader is directed to the excellent reviews by Dasgupta and Jeong [3] and Lopes [4]. However, here, we will attempt to highlight elements of what is known about the embryological and particularly molecular pathways involved in meninges development as it may relate to the development of meningiomas and the presence of meningothelial tissues in other anatomical locations outside of the central nervous system (CNS) proper.

In contrast to other areas of CNS development, relatively little is known about the molecular characteristics of meninges development. In a broad sense, however, the meninges increasingly are being shown to be critical to proper calvarial and underlying brain development. It is noted that the cranial meninges are derived from two cellular pools; the neural crest (ectoderm) and mesoderm. Neural crest derived cells can be found in the three layers of meninges covering the forebrain cerebral hemispheres but not in the meninges covering midbrain or hindbrain. Calvarial development closely parallels meninges development in that the frontal bones are primarily neural crest derived while the parietal bones are of mesodermal origin. Foxc1 is a key and ubiquitously expressed transcription factor in meninges development with noted early upregulation in the primary meninx. Its prominent role is demonstrated by lack of apical arachnoid and dura mater formation as well as lack of apical calvarial development in Foxc1 mutant mice. Parietal bone development appears to depend on the underlying meninges (derived from neural crest) expressing transforming growth factor beta 2 receptor (Tgfbr2) as Tgfbr2 mutant mice show severe defects in both parietal bone and underlying meninges development. The dura or outer dense layer of the meninges becomes closely apposed to the underside of the bony calvarium and is the de facto periosteal layer. The mesenchyme around the developing brain is divided into layers that include a dermal layer (dermis of scalp), skeletogenic layer (skull), and then the meningeal primordium. By extension, it can be plausible that ectopic meningiomas can arise (and indeed do) in the bones of the calvarium and scalp. It has been proposed that arachnoid cells or precursors can migrate with cranial nerves as they exit their foramina during development and be the forerunners of primary ectopic meningiomas in anatomic regions such as the orbit, ear, and neck [1]. Meninges appear to play crucial roles in brain development including providing trophic factors for neuronal survival, migration/positioning of neurons, neuronal generation from neuronal progenitors, blood vessel development, corpus callosum development, and may provide a niche for neural stem cells [5].

Again, by extension, it is not implausible to think that since much of the structures comprising the head and neck are derived from neural crest (and mesenchymal) cells, cells with multipotent capabilities, that some of these cells could be dormant and later directed down a path of meningeal differentiation in aberrant locations.


**3. Indications**

The first ever use of an optical tool to visualize the interior of the human body was performed by Bozzini in 1806 [1]. A hundred years later, Lespinasse used a cystoscope to visualize the ventricles of two hydrocephalic children [2]. In 1918, Dandy performed an endoscopic avulsion of the choroid plexus in five hydrocephalic children (where four died) [3]; he called his instrument a "ventriculoscope." In 1922, he opened the floor of the third ventricle by sacrificing an optic nerve [3, 4]. In 1923, Mixter performed a third ventriculocisternostomy through the anterior fontanelle [5], which is considered the first ever successful ETV. In 1947, McNickle was the first to introduce a percutaneous method of performing the third ventriculostomy that led to decline of the complication rate, thus improving the success rate [6]. Afterward the endoscopic technique progressively developed to reach the current modifications in the ventriculoscope for better, clear, and safe visualization of the intraventricular

There is still a detectable failure rate of all treatment modalities of hydrocephalus. However, ETV represents a convenient and easy mode of management. In the recent studies that evaluated the endoscopic third ventriculostomies performed for the treatment of obstructive hydrocephalus, success rates were found between 50 and 94% [8–11]. The type of hydrocephalus and age of the patient, in addition to the surgical technique, play an important role in the success of the ETV [11]. We will discuss comprehensively the surgical technique, indications, and current challenges regarding the increase of the success rate of

A preoperative MRI is almost always needed prior to surgery. From the frontal coronal burr hole, one reaches first the central part of the lateral ventricle near the frontal horn. The frontal horn is demarcated by the absence of choroid plexus. The lateral wall is formed by the nucleus with subependymal veins; medially is the septum pellucidum with septal veins. The choroid plexus and the foramen of Monro are very important landmarks for the central part of the lateral ventricle. The plexus is situated in the floor of the lateral ventricle, the thalamostriate vein lies laterally, and the septal vein's meeting point is on the medial wall; these three structures form the Y-shaped configuration necessary for orientation. The foramen of Monro is formed anterolaterally by the fornix, posteromedially by the anterior thalamic tubercle (**Figure 1**). On looking backwards with the endoscope, the body of the lateral ventricle back to the region of the trigone, with the body of the caudate laterally underlying the thalamostriate vein and the stria terminalis thalami. Adequate orientation of the morbid anatomy that can occur due to

By entering the foramen of Monro, the floor of the anterior part of the third ventricle is clearly identifiable, with the mammillary bodies and tuber cinereum as the two main structures

Liliequist's membrane is an arachnoid leaflet situated in the basal cisterns and is a very impor-

anatomical structures [7].

96 Hydrocephalus: Water on the Brain

**2. Endoscopic anatomy**

chronic hydrocephalus is needed [12–14].

needed for the anatomical orientation (**Figure 1**) [14].

tant anatomical structure in the postsellar region.

the ETV.

ETV is considered the first management option in adult patients with obstructive hydrocephalus by many neurosurgeons. It is a reliable management option in adults with aqueductal stenosis with a success rate that reach 88% [15]. Stenosis at the aqueduct of Sylvius can be congenital or acquired. In three quarters of cases, the root cause might be unknown [16]. It is not considered as a stable condition as it can be tolerated for years, where stenosis is aggravated by trauma, subarachnoid hemorrhage, viral infection, or gradual accumulation of the CSF proximal to the aqueduct in partial obstruction [16–18]. In a previous study, clinical improvement with identifiable success of the procedure was detected in 86.4% of cases [19], where the success rate was lower in secondary ETV after VP shunt (**Figure 2**). This would be better identified in patients with previous history of multiple VP shunt revisions where the ETV failure is relatively more encountered [19, 20]. ETV is also preferred as the first-line management of hydrocephalus due to obstruction of the aqueduct of Sylvius with pineal tumors or tectal gliomas [21–23].

ETV is less successful in pediatric age groups, with the lowest success rate in children younger than 6 months of age, even in aqueductal stenosis [24, 25].

The application of the ETV has been expanded to patients with hydrocephalus associated with fourth ventricular outlet obstruction, Dandy-Walker malformation [26], Chiari malformation [27–29], communicating hydrocephalus [30], and normal pressure hydrocephalus [31, 32].

widening, with subsequent opening of the liliquist membrane, is important for a direct visualization of a naked basilar artery (BA) [25]. A delicate surgical technique is required with experienced hands during the opening of the floor of the third ventricle till the BA is clearly

Endoscopic Third Ventriculostomy, Indications and Challenges

http://dx.doi.org/10.5772/intechopen.73518

99

The ETV Success Score (ETVSS) has been developed and validated to predict ETV success based on certain variables [34, 35]. It depends on predicting the success according to the age of the patient, cause of hydrocephalus, and presence of the previous shunt operation. The success rate can be predicted according to these variables. However, intraoperative factors "like the presence of excessive adhesions, mobility of the stoma, excessive bleeding, and opening of Liliequist's membrane" (**Figure 3**) should be taken into consideration in predicting the success of ETV [36]. In addition, the VP shunt independence is considered a generalized but competent method to measure the success of ETV after VP shunt failure

The change of the ventricular size with a deterioration of the clinical condition has been well known as one of the signs that identify hydrocephalus. In addition, the decrease of the ventricular size after management which accompanied improvement of the general condition has its additional value of success confirmation (**Figure 2**). However, the change of the ventricular size is not well supported as an accurate measurement of the effective treatment of hydrocephalus, especially when it is irrelevant to the clinical condition of the

Specific intraoperative factors are considered significant in addition to the associated morbidity. This would include the duration of surgery, type(s) of endoscope used, and degree of

**Figure 3.** Another patient during ETV where thick arachnoid membranes (arrow) surrounding the basilar artery (b)

visualized to avoid major vascular injury [33].

**5.1. Clinical evaluation and radiological evaluation**

**5. Outcome**

[37, 38].

patient [39].

intraoperative bleeding [33, 36].

denoting possibility of ETV failure.

**Figure 2.** CT of the brain of a previously shunted 12-year-old male with aqueductal stenosis that had signs of increased tension and VP shunt failure; the CT shows enlarged ventricles (A) where ETV was performed without removing the shunt. Follow-up CT of the brain 3 months afterward showed decline in the ventricular size (B) which was accompanied by clinical improvement of the patient.

However, in exclusion to adult-type obstructive hydrocephalus, there is still a lack of strong evidence that supports the procedure.


**4. Time of rejection of the corneal implant**

According to the evidence of rejection time after surgery the rejection must be classified in hyperacute, acute or chronic rejection. The hyperacute rejection occurs within days after transplantation of the graft. This type of rejection has become rare, affecting less than 1% of transplant recipients due to improved pre-transplant projections. Hyperacute rejection occurs when the host antibodies recognize and bind to antigens of the graft (such as ABO blood group proteins or proteins of major histocompatibility complex). The binding of these antibodies lead to the initiation of the complement cascade, neutrophil recruitment, platelet activation, endothelial cell damage of the graft, and stimulation of coagulation reactions, which in turn lead to rapid thrombosis, loss of vascular integrity, heart tissue, and loss of graft function.

The acute rejection occurs in approximately fifty percent of transplant recipients experience acute rejection (with only 10% progressing to graft loss), which can occur several hours or days (even weeks) after transplantation. The incidence of acute rejection has decreased significantly with the successful use of immunosuppressants such as cyclosporine and azathioprine. The incidence of graft loss was reduced by the latest anti-rejection treatments. Acute rejection occurs when alloantigen-reactive T cells from the host to infiltrate the graft and become activated by contact with foreign proteins, related to the graft presented to

The Complications After Keratoplasty 115

make up for esthetical reasons around the eye, perhaps it can interfere the microbial equilibrium of normal flora and let the opportunistic flora to colonize the eye and cause infections. Among some aftercare corneal transplant recommendations after this finding

Prevention of rejection (immunological exams before surgery donor and receptor)

10-0. And the use of an antibiotic ointment administered at the end of surgery.

restriction recommended by American Ophthalmology Association:

glasses during the waking day, for 1 month.

slightly backwards for 2 weeks.

8. No swimming for 3 weeks.

10. Walking is permitted.

6. No heavy exercise of any kind for 3 weeks 7. No sexual intercourse for 3 days after surgery.

Other important recommendations to verify are:

9. Not read for more than 10 minutes at a time for 2 weeks.

5. May watch TV

To prevent post surgical infections the disinfection protocols are the main factor to consider for minimize complications: before starting the surgical procedures, all of the recipient eyes (including eyelids and conjunctiva) can be rinsed with povidone iodine solution, 5%, that was allowed to act for 3 to 5 minutes. After drying the periocular surface, the operation field was covered with sterile drapes and PK can be performed as follows: rinsed with sterile solution (balanced salt solution, it is recommended to introduce acetylcholine chloride (like Miochol-E) into the anterior chamber. Prior to donor trephination and the graft's sutural fixation, the donor's endothelium was covered by sodium hyaluronate, 1% (Healon ophthalmic viscosurgical device). To fix the grafts, a double-running cross-stitch suture with

It is important also instruct patients avoid restrictions and talk about postsurgical care to prevent fails. Some of these are: To take de prescription of antibiotics and drugs, and the

1. Use metal shield nightly or when taking a nap during the day and a cloth pad under

Recruitment of donor tissue : de donor must be removed within six hour after death, the viable storage period of the removed cornea-scleral button is two weeks, grafts donors < 12 months or > 70 years are preferably not to be used and for more security it is also important not use corneas from death of unknown causes, certain infectious diseases like Jacob-Creutzfeld, SSPE, progressive multifocal, leuko-encephalopathy (CNS), certain systemic infection (AIDS, septicemia, syphilis, viral hepatitis), leukemia and disseminated lymphoma, intrinsic eye diseases (tumors, active inflammations, previous intra-ocular surgery), with respect to the recipient cornea : absence of corneal sensations, stromal vascularization, corneal thinning at the expected recipient-donor margin, active inflammation) and also it is so important to verify the surgical procedure : decide about graft size, usually graft size is no bigger than 8,5 mm in diameter to avoid post-keratoplasty increase in intra-ocular pressure, anterior synechiae and

2. Not bend at the waist for more than 10 minutes at a time, but may squat at the knees. 3. Not lift or push anything heavier than 15 pounds, including grandchildren, for 2 weeks. 4. Hair may be gently shampooed by a friend or a beauty shop with the head leaning

around the world it should look the next factors:

More research for therapy protocols

 Integrity of sutures Ephitelization

Non-filtration.

them by antigen presenting cells. These T cells can lead to tissue damage of the graft by the direct elimination of graft cells (killer T cells) or the production of proinflammatory cytokines such as tumor necrosis factor, interleukin-1, and interferon. These cytokines are vasoactive and perpetuate the inflammatory cell recruitment and infiltration. As a result, graft inflammation progresses, leading to tissue distortion, vascular insufficiency, and cell destruction - all of which may eventually compromise graft function.

By the other hand, the chronic rejection occurs in 50% of transplant patients within 10 years after transplantation. This form of rejection is characterized by the development of occlusion of blood vessels luminal progressive thickening of the intima of medium and large artery walls. Chronic rejection is a pathological response of the tissue remodeling that takes place at a variable rate after graft endothelial cells are traumatized by mechanical damage, ischemia, and immune system during and after the transplant. Damaged vascular endothelial cells produce cytokines and tissue growth factors that initiate vascular repair, causing the underlying smooth muscle cells to begin proliferating. Large amounts of intimal matrix occur, leading to vascular wall thickening stop growing. Slowly progressive reduction in blood flow, results in regional tissue ischemia, cell death and tissue fibrosis.


**5. Conclusion**

The state of Assam, popularly known as the land of the red river and blue hills is home to a diverse array of flora and fauna. Assam falls in one of the great migration routes of mankind of different groups who over the centuries have come and settled down. Every community has its own traditional rituals, customs and herbal remedies which have been molded by the geographical location and the environmental factors where they reside. The abundant natural resources in encompassing location form the basis for the characteristic food habits and related medicinal practices of each community. By their experience, the knowledge of herbal remedies was transferred to generation after generation as folk medicine.

A study was conceived based on the aforesaid facts with intent to scientifically analyze different folkloric healing practices encompassing various medicinal plants. Subsequently an ethno medicinal survey was conducted across the state of Assam for compiling information with respect to traditional medicine. Thereafter, plants belonging to Costaceae family were selected for scientific validation studies owing to their predominant use among the traditional healers in the surveyed regions particularly in upper Assam for treating ailments like jaundice, diabetes etc.

Three plants belonging to the costus genus were identified *viz. Costus scaber, Costus speciosus* and *Costus pictus* for the study. *Costus speciosus* locally known as 'Jomlakhuti' in Dibrugarh, Golaghat and Tinsukia district; 'Peki jigjig' in Dhemaji; 'Ai-upo'in Karbi Anglong district and 'Buritokon' in Kokrajhar district, the rhizomes, leaves are primarily used for treating liver aliments, diabetes, UTI, snake bite respectively. *Costus scaber* locally known as 'Keuri' in Dhemaji district, the leaves are used in the treatment of snake bite and wound healing. *Costus pictus* locally known as 'Leteki' in Golaghat and Tinsukia district and 'Tui' in Karbi Anglong district, the aerial parts and leaves are used traditionally in the treatment of diabetes, for blood purification and jaundice respectively.

Therefore, it can be safely concluded that species belonging to this genus are traditionally used in the mitigation of various ailments particularly diabetes. Furthermore, *in vivo* and *in vitro* studies are warrented against these species so as to elucidate viable phyto components as a future prespective.

#### **Acknowledgements**

The authors thankfully acknowledges the traditional healers of Dotma, Kokrajhar district; Dhupdhora, Goalpara district; Laipuli, Tinsukia district; Kathkatia village of Silonijan of Karbi Anglong district; Naojan and Baragharia village of Golaghat district; Nagakhelia village and Jokai area of Dibrugarh district; Sissiborgaon, Barmukuli and Majarbari village of Dhemaji district of Assam who helped by sharing their valuable information regarding the methodology of usage of different plant species used in the treatment of ailments. The authors also acknowledge Mrs. Monika Kuli of Barmukuli village of Dhemaji district, Mrs. Sarala Rabha of Dhupdhora of Goalpara district, Mrs. Minu Borah and Mr. Dhruba Borah of Baragharia village and Mrs. Purnima Borah of Jyotinagar, Golaghat district, Mrs. Savitri Sonowal of Jokai and Mr. Anil Bhuyan and Mr. Ripul Bhuyan of Nagakhelia village of Dibrugarh district, Mrs. Kareng Rongpi of Silonijan of Karbi Anglong district, Dr. Pranjit Narzaree, Ms. P. Narzaree of Kokrajhar district for their immense help regarding the collection of information in the conducted ethnomedicinal survey. The conducted study was not funded by any organization whether government, semi government or private funding bodies whatsoever.


Preface

One of the most interesting and challenging fields of medicine and surgery is organ donation and transplantation. It is a field that has made tremendous strides during the last few decades through the combined input and efforts of scientists from various specialties including surgeons, hepatologists, nephrologists, immunologists, ethicists, and infectious disease specialists. What started as a dream of pioneers in the field has become a reality for the thousands of patients whose lives can now be saved and improved. However, challenges remain significant, as do the expectations, for what was once an experimental treatment could eventually become the future of medicine because it involves most organs including the heart, lungs, eyes, liver, kidneys, pancreas, and small intestine.

Written by an excellent group of world authorities, this book presents the challenges in the field of organ donation and transplantation, including ethical, legal, and medical issues, as well as the technical and immunological problems facing experts involved in the care of transplant patients. In addition to the knowledge shared, the authors provide their personal clinical experience, making this book an extremely useful tool for every scientist and physician practicing in the field of transplantation.

The book is divided into three sections on organ donation and transplantation. The chapters include information on the current state and different types of donation, the challenges identified in increasing donations as well as potential solutions.

This book is a worthy contribution to the literature on organ donation and transplantation. It is designed for scientists, physicians, and surgeons across various specialties who are invested in improving the lives of transplant patients.

> **Georgios Tsoulfas, MD, Ph.D., FICS, FACS** Professor of Transplantation Surgery, Chief Department of Transplantation Surgery, Aristotle University of Thessaloniki School of Medicine, Thessaloniki, Greece

**1**

Section 1

Organization of Organ

Donation

### Section 1