Tuberculosis (TB) is an airborne contagious bacterial disease, which ranks as the second leading cause of death from an infectious disease worldwide, after HIV. According to the 2012 World Health Organization [18]global TB report (http://apps.who.int/iris/bitstream/10665/75938/1/9789241564502_eng.pdf), in the year 2011 itself 8.7 million people fell ill with TB while 1.4 million died due to the disease [19]. Lack of adequate diagnostic measures for timely detection were the main concern preventing proper response to tackle the morbidity and mortality, especially in the HIV associated and drug resistant TB cases. In low income and high incidence countries, diagnosis is still dependent on traditional techniques such as sputum smear light microscopy and sometimes culture. However, these microscopy techniques are far less sensitive ranging from 20 to 80 percent sensitivity [20]especially in HIV patients and children where pulmonary bacillary load is less than detection limits of microscopy [21]. To improve sensitivity, WHO has recommended use of light emitting diode [22]microscopy which can generate both light and fluorescence wavelength instead of conventional light or fluorescence microscopes [23]. Little Improvement in microscopy technique was still not appropriate to tackle diagnostic challenges such as HIV co-infected patients and drug resistance cases, which made cultivation indispensable. According to the WHO guidelines, microscopy negative HIV patients with TB symptoms are to be tested by culture as well (Table 1).

Multidrug resistant TB, broadly categorize as MDR-TB (mainly resistant to INH and RIF) and XDR-TB (Resistant to additional antibiotics) are the major concerns for the need of rapid and effective diagnostic techniques which is traditionally identified by conventional culture and drug susceptibility test (DST). Culture techniques in the resource poor countries are inefficient due to lack of infrastructure, poor biosafety measures as well as unavailability of trained staff to perform reliable tests. Moreover, crucial time is lost during cultivation and DST. Various immunological techniques like serologic test and Enzyme-linked immunosorbent assay (ELISA) were also tested for the TB diagnosis but were not successful due to low sensitivity, and cross reactivity [24][25]. More advance techniques like DNA based molecular line probe assays [26]have been introduced based on the genetic studies suggesting that the drug resistance in certain strains is due to mutation at the drug target site [27][28][29]. Line probe assays [26]is a Polymerase chain reaction (PCR) based reverse hybridization molecular drug susceptibility assay which is very specific (>99%), sensitive (>97%) and

Abbreviations: Light emitted diode [22], ELISA (enzyme-linked immunosorbent assay), Rapid detection test [26], Line Probe Assay [26], Real Time Polymerase chain reaction (RT PCR).

rapid and does not require viable pathogen for the detection which makes handling and biosafety more convenient. However, LPA probes are mainly specific for MDR-TB but not extensively drug-resistant tuberculosis (XDR-TB) since no single mutation is responsible for extensive drug resistance [29][30]. Therefore, advent of LPA did not eliminate the need of conventional cultivation especially for the diagnosis of XDR-TB. In 2009 WHO endorsed LPA coupled with liquid media cultivation technique for TB diagnosis in endemic countries [23](Table 1).

Real time PCR (RTPCR) is one of the advance and rapid DNA based method, which amplifies DNA in a closed system and gives DNA melting profiles to detect resistance associated mutation. One of the fully automated real time PCR based technique named Xpert MTB/RIF can detect TB, and identify rifampicin resistance directly from sputum under two hour [31]. Clinical validation of the Xpert MTB/RIF technique suggested 100% specificity for smear positive culture positive as well culture negative cases [31]. Xpert MTB/RIF system has offered excellent detection performance with lower biosafety requirements and ease of equipment operation. Compact real time PCR Xpert MTB/RIF system is easy to transport and thus can provide onsite diagnosis at point of care to the patients. The major limitation of the Xpert MTB/RIF method is the high cost of reagents and instrument compare to LPA or other assays. Since majority of the drug-resistance cases are rifamycin therefore WHO has endorsed the Xpert technology in 2010, and is monitoring the global roll out of the technology to promote effective coordination [32]. The TB-Xpert Project will provide approximately 1.4 million Xpert MTB/RIF test cartridges and over 200 GeneXpert instruments for the rapid detection of TB and rifampicin resistance in 21 South East Asian and African endemic countries from year 2013 to 2015 [19]. However, Xpert MTB/RIF technology does not eliminate the need for conventional microscopy culture and DST, which are required to monitor treatment progress and to detect resistance to drugs other than rifampicin. In settings or patient groups where rifampicin resistance is rare, Xpert MTB/RIF results indicating rifampicin resistance should be confirmed by conventional DST or LPA. One of the most promising and upcoming diagnostic technique is the DNA microarray chip platform which can detects all the gene mutations simultaneously to target any drug resistance [33][34](Table 1). Microarray technique can perform identification, genotyping as well as drug resistance due to every known mutation in one experiment simultaneously. Equipped with immense potential; microarray technique is still at the stage of infancy and would require lots of optimization and clinical trials before it becomes a standard diagnostic technique for TB.

Looking at the overall scenario and present challenges no single technique is the gold standard for TB diagnosis. Therefore, an integrated tiered level approach is advisable where the diagnosis of the disease is performed in the laboratories at different levels. Each level is divided based on the complexity and availability of the resource and trained personnel. The very first level should be onsite or can be in the rural area where simple microscopy and sample collection can be performed. Next level should be the laboratories where samples can be transferred to perform better microscopy, conventional culture and DST tests with adequate measures. Final level should be sophisticated hi-tech laboratories headquarters, which can perform genotyping, further drug resistance test and research for better diagnosis and cure. Coordination between each level of the laboratories is the most important step towards successful management of the disease.

Malaria is one of the most prevalent and deadly parasitic diseases especially in the underdeveloped countries of Africa and South East Asia. According to the latest report from WHO there were about 219 million estimated cases of malaria in 2010 (with an uncertainty range of 154 million to 289 million) and 660,000 deaths (with an uncertainty range of 490,000 to 836,000) [18]. In the year 2012, new initiative from WHO global program T3 (Test, Treat and Track) developed to provide universal access to diagnostics, treatment and stronger surveillance. Early diagnosis is important for the proper treatment, and control of transmission of disease. Most evaluated and successful techniques for Malaria diagnosis so far are Giemsa microscopy and rapid diagnostic tests (RDTs) [35][36]. Due to its lower cost and simplicity, giemsa staining microscopy still remains the standard method for rapid detection of parasite in rural endemic area [37](Table 2). However, Low sensitivity (50 - 100 parasite per µl), false positive results and emerging complications in the diagnosis like dealing with 4-aminoquinolines drug resistance P. falciparum strain and low level of infection make conventional techniques inadequate for the purpose. In the last few decades of malaria research alternative methods like ELISA [38], Immunofluorescence assay [39], RDTs [40][41]and recently DNA based assays have been introduced. Among them, so far RDTs have shown promising results due to its similar sensitivity to microscopy (200 parasites per μl in clinical settings) but ease of use with no instrumentation or technical skill requirement and point of care (POC) availability. In 2006, WHO, Special Program for Research and Training in Tropical Diseases [42]and the Foundation for Innovative New Diagnostics (FIND) launched an evaluation program to assess the comparative performance of commercially available malaria RDTs. SO far four rounds of testing have been performed on 164 RDT products and published [36]. P. falciparum tests targeting HRP2 antigen demonstrated the highest PDS however tests targeting pLDH for P. falciparum and P. vivax detection did not pass round 1 (<80% PDS for P. falciparum at 2000 parasites/μl). The results of the worldwide RDT evaluation program would further guide policy makers of government agencies towards deciding better-performing tests.

Non-sensitivity for all Plasmodium species, thermosensitivity, inability to detect low level of infection (less than 200 parasites per μl), and false positive results are the major concerns for RDTs at this point for efficacy of these standard diagnostic measure. DNA based diagnostic techniques have advantage of being more sensitive, specific, determining species, drug resistance and low level of infection. PCR is one of the basic and sensitive DNA based technique and has limit of detection up to 0.5 - 5 parasites/ml [43][44]. Isothermal amplification methods such as Loop mediated isothermal amplification (LAMP) is widely

Abbreviations: Rapid detection test [26], ELISA (enzyme-linked immunosorbent assay), Ligation Detection Reaction (PCR-LDR), Ligase Detection Reaction?Fluorescent Microsphere Assay (LDR-FMA), Loop mediated isothermal amplification (LAMP).

studied DNA based amplification technique in Malaria diagnosis, which does not require thermos-cycler and has 95% sensitivity, and 99% specificity with documented detection limit of 0.2 parasite/ml [45][46]. Other DNA based techniques such as real-time PCR, Multiplex PCR/Ligation Detection Reaction (PCR-LDR), and Ligase Detection Reaction?Fluorescent Microsphere Assay (LDR-FMA) have also been introduced and tested (Table 2). Major drawback of the DNA amplification techniques are expensive reagents, instrument requirements and special care in handling of samples as they are prone to contamination and amplification of non-targeted DNA sequences. Currently, these techniques are limited to high profile lab or central health care facilities due to their resource intense requirements and high cost. Novel strategies are needed to further research to improve and incorporate these techniques into routine health centers in endemic areas.

Overall, in the present scenario both low technology and high technology approaches are indispensable for successful parasite detection towards management and eventually in the eradication of the disease. RDTs and microscopy are suitable for the majority of symptomatic P. falciparum detection and management while molecular based advance techniques are required for detection of low level of infection and asymptomatic individuals who may contribute to continuing malaria transmission and P. vivax cases.

AIDS, caused by HIV is the major public health issue in the world. According to a recent WHO and UNAIDS data, 36.9 million people were living with HIV globally at the end of 2014 while 1.2 million people died and 2 million newly infected [47]. Sub-Saharan Africa is the most affected region accounting for almost 70% of global HIV infection. There is no cure for HIV. However, timely detection the HIV status can be beneficial for effective antiretroviral therapy (ART) for productive lifestyle and preventing the spread of the disease. In present days, there are three types of popular HIV diagnostic tests available including antibody tests like ELISA, rapid test or Western blot [48], antigen/antibody combination tests like viral protein p24 along with HIV antibody [49][50][51], and nucleic acid tests (NAT) [52][53](Table 3). Antibody tests detect antibodies, proteins that the body makes against HIV, not HIV itself. Antigen tests and RNA tests detect HIV directly. Fourth generation techniques for detection of antibody and antigen simultaneously can reduce the time of diagnostic window after primary infection compared to antibody alone [54]. Nucleic acid amplification test (NAAT) mainly rely on amplification of the nucleic acid by PCR and can be qualitative as well as quantitative. PCR assays have become more popular nowadays due to its sensitivity and ease of technique [55]. Since the advent of human immunodeficiency virus [26]testing, laboratory based methods have undergone tremendous change. Western blot and indirect immunofluorescence assay [56]have been excluded in the updated CDC recommendations due to false negative results [57][58][59]. An initial HIV antibody test or antigen/antibody test is performed along with some more follow-up confirmatory testing as per the updated Centers for Disease Control and prevention [36]and WHO guidelines [60]. Based on updated CDC guidelines (Laboratory Testing for the Diagnosis of HIV Infection: Updated Recommendations Published June 27, 2014, https://stacks.cdc.gov/view/cdc/23447), laboratory personnel should use Food and Drug Administration (FDA) approved assays for the diagnosis of HIV infection in adults and children > 24 months of age. Testing should be performed with ag/ab detection tests, a combination immunoassay that detects HIV1 and HIV2 antibodies. All positive specimens on this initial assay should undergo further testing with an immunoassay that differentiates HIV-1 from HIV-2 antibodies. Specimens that are reactive on the initial immunoassay and non-reactive or indeterminate on the antibody differentiation assay proceed to HIV-1 nucleic acid testing for resolution, which looks for the virus RNA directly. Positive results from the recommended algorithm indicate the need for HIV medical care, and an initial evaluation that includes additional laboratory tests (such as HIV-1 viral load, soluble cells of differentiation [sCD4+], T lymphocyte determination, and antiretroviral resistance assay) to confirm the presence of HIV-1 infection. It is used further to stage HIV disease, and to assist in the selection of an initial antiretroviral drug regimen (OARAC, Panel on Antiretroviral Guidelines for Adults and Adolescents). Guidelines for the use of antiretroviral agents in HIV1-infected adults and adolescents is available electronically at (http://www.aidsinfo.nih.gov/ContentFiles/AdultandAdolescentGL.pdf last

Abbreviations: Nucleic acid amplification test (NAAT).

updated October 2017. In the 2012 Geneva meeting, WHO has recommended multitier approach to diagnose and treat HIV in epidemic and non-epidemic areas especially in the developing countries based on the resource availability (WHO Expert Meeting Report Geneva, Switzerland, 6 - 7 June 2012: http://apps.who.int/iris/bitstream/10665/75971/1/9789241504522_eng.pdf).

Level 0: Community outreach setting: Community health worker for spreading awareness, HIV RDTs (Rapid diagnostic tests).

Level 1: Primary care setting: trained health care workers: nurses, clinical officers HIV RDTs, other POC tests, database collection.

Level 2: District: Laboratory technicians and assistants EIA for diagnosis, low throughput soluble CD4 (sCD4+), chemistry, hematology, microbiology.

Level 3: Regional or provincial: Laboratory specialists/senior technicians EIA for diagnosis, higher throughput sCD4, HIV molecular technologies including HIV VL, quantitative/qualitative “Early infancy detection” (EID).

Level 4: National: Senior laboratory specialists using enzyme immunoassays (EIA) for diagnosis, higher throughput sCD4, HIV molecular technologies including HIV viral load (VL), quantitative/qualitative EID, HIV resistance testing.