Pneumocystis jirovecii is an extracellular organism that causes pneumocystis pneumonia. It is usually found colonizing the alveolar spaces of the lungs although extra pulmonary manifestations have been reported.

Currently, the documented frequency of pneumocystis infection is increasing in Africa, with PCP detection in up to 80% of infants with pneumonia [1] .

A limited number of epidemiological studies have evaluated the significance of PCP in developing countries compared to developed countries where the epidemiology and clinical picture of PCP is clearly well defined and documented [2] . Recent reports indicate an increased rate of PCP in Africa, Asia, and South America [3] . Mortality rates of 30% - 50% have been documented in several studies [4] . In persons with immunosuppressive underlying conditions, PCP carries worse prognosis and this has not changed in the past 20 years. In tuberculosis (TB) endemic parts of the world, substantial numbers of patients with pulmonary symptoms are managed as “smear negative TB patients” or treated for TB without a definitive diagnostic criterion [5] [6] . In sub-Saharan Africa, tuberculosis could be a common co-infection in persons with PCP [7] . However, the lack of access to routine PCP diagnosis is the limiting factor. The study sought to determine prevalence of Pneumocystis jirovecii Pneumonia (PCP) and compare specificity and sensitivity of Toluidine staining method and Polymerase Chain Reaction (PCR) in sputum of TB smear negative and retreatment patients at Coast General Hospital (CGH).

Despite HAART medication PCP continues to be a deadly illness in AIDS patients. Previously, the prevalence of PCP among AIDS patients in Sub-Saharan Africa was unknown [13] . Recent data from Africa suggests an underreported opportunistic infection among AIDS patient. Recent research suggested that PCP may be more common in TB Patients with negative smears. The use of cotrimoxazole in HIV/AIDS patients may have impacted the prevalence of prevalence of PCP in Africa [1] [12] [13] .

Out of the 100 patients, 63 men and 37 women. Patients aged 31 to 53 made up 62% of the patients (Min = 11y, Max = 85y). The median age 42 years. 14 females and 27 males had the Pneumocystis Jirovecii Pneumonia. Majority of patients—35.8%—used charcoal (n = 29); 17.3% charcoal + firewood, kerosene, or gas (n = 14). 17.3% using kerosene (n = 14), 16% firewood (n = 13), and 13.6% gas (n = 11) for cooking. 19 patients on TB treatment i.e. Table 1 , Table 2 and Table 3 . Cases worsening or new cases TB treatment. 81 patients not currently on treatment but referred. Patients negative on TB diagnostic algorithms with PTB like symptoms are often considered TB relapse and retreated. As shown in Table 4 .

Toluidine blue 0 stains also apparently stained only the cyst forms. Results with the TBO are shown in Figure 1 . The cysts are lavender in color but are somewhat grainy in appearance; much background material is also evident. In Figure 1 structures thought to be P. jirovecii cysts have a spherical appearance and the outlines of individual cysts are quite faint. The cyst forms appear as lavender structures approximately 5 µm in diameter, and many are Pneumocystis organisms have a high tropism for the lung [14] [15] .

The life cycle of Pneumocystis mainly consists of 3 major life cycle stages identified by morphology; the cyst, the pre-cystic and the trophic form stages. The trophic form is mono nuclear and thin walled and usually dominates during active infection and fills the alveoli space and is variable in length 1 to 8 µm long with multiple pseudo hyphal structures which are used as an anchor to host cells [16] . The alcohol and xylene washing procedures remove excess toluidine blue O from the slide. In some other cases, P. jirovecii was difficult to detect because of the presence of background debris. P. jirovecii cysts typically occur both singly and in small clusters of closely associated organisms [17] .

A total of 41 samples out of the total 100 expressed the 346-band fragment on the agarose gel. Nested PCR prevalence of 41% (41 cases) TBO staining lower prevalence of 29% (29 cases). TBO had 71 negative cases, PCR 59 negative cases as in Table 5 and Table 6 . Males’ seemingly high prevalence susceptibility to TB could have been attributed to their poor health seeking behaviors [18] .

The mitochondrial Ribosomal RNA large subunit gene is currently used as a PCR target for PCP diagnosis [9] [17] . According to a study done by Nowaseb et al., 2014, P. jirovecii was detected in 25/475 (5.3%) patients. Seventeen patients (3.6%) tested positive for P. Jirovecii by qPCR and direct microscopy Eight patients (1.7%) were positive only by qPCR. None of the samples tested were positive by direct microscopy alone [19] .

Mt LSU-rRNA, is one of the highly sensitive and specific genetic regions because they are multiple copies of genomes in it [19] . The Mt LSU-rRNA gene is involved in basic metabolic functions and is considered to be a highly informative locus. This is also the locus used most widely for PCR-based detection of P. jirovecii, as it is present in a large number of copies [20] .

Nested PCR additional 12 patients than TBO technique-high sensitivity, TBO had acceptable sensitivity & very high specificity ( Table 6 ). In developing countries rates of PJP are lower, possibly due to poor survival, under diagnosis, prior infection with tuberculosis and other organisms [21] - [23] .

However, locally no studies have been done to determine the significance of co-infection of Pneumocystis pneumonia with tuberculosis [24] [25] .

Given that most tuberculosis patients are co-infected with HIV/AIDS and are severely immunocompromised [26] [27] , there is a possibility that Pneumocystis pneumonia is a significant co-pathogen in this group of patients [28] [29] .

Pneumocystis pneumonia has been shown to be a co-infecting pathogen with other respiratory pathogens with a prevalence of [30] - [32] .

However the majority, 89% of this study specimens were negative for P. jirovecii, These findings highlight the neccesity and the importance of fungal diagnosis in high risk population especially those with pulmonary TB [33] [34] . According to this study chi-squire test, there was no significance of co-infection Pneumocystis jirovecii with Mycobacterium tuberculosis with p-value of 0.86 [35] [36] . In order to evaluate the sensitivity and specificity of Nested PCR and Toluidine Blue O staining method in identifying P. jirovecii in the sputum of patients [37] [38] with tuberculosis smear negative and retreatment cases with the nested PCR method serving as the gold standard as shown in Table 7 .

The sensitivity of toluidine blue O staining is 33.82%, which indicates that it correctly identifies 33.82% The specificity of toluidine blue O staining is 97.82%, indicating that it has a high ability to correctly identify true negative cases compared to PCR, suggesting that it is good at ruling out non-P. jirovecii cases.

The sensitivity of toluidine blue O staining is 33.82%, which indicates that it correctly identifies 33.82% of true positive cases compared to PCR, missing a significant proportion of true positive cases. The specificity of toluidine blue O staining is 97.82%, indicating that it has a high ability to correctly identify true negative cases compared to PCR, suggesting that it is good at ruling out non-P. jirovecii cases [39] - [42] .

The Positive Predictive Value (PPV) of toluidine blue O staining is 95.83%, indicating that when it gives a positive result, there is a 95.83% chance that the patient actually has P. jirovecii according to PCR. This suggests that a positive result with toluidine blue O staining is highly likely to be accurate ( Table 7 ).

The Negative Predictive Value (NPV) of toluidine blue O staining is 78.95%, indicating that when it gives a negative result, there is a 78.95% chance that the patient truly does not have P. jirovecii according to PCR ( Table 7 ). A negative result with toluidine blue O staining, however, should be interpreted with caution, as it has a relatively higher rate of false negatives. The accuracy of toluidine blue O staining is 83.95%, which indicates the overall proportion of correct results (true positives + true negatives) compared to PCR [32] .

A positive TBO stain usually indicated PCP, so this may be considered the minimum frequency of infection (3.6%) [43] [44] . Simultaneous co-contamination with M. tuberculosis and P. jirovecii isn’t always thought to be common, which may explain why the prevalence of P. jirovecii appeared to be low in regions with high M. tuberculosis burdens [45] [46] . The presence of PCP might also suggest colonization of those patients; however, it became more likely that Candida became a co-present oral pathogen in sufferers with PCP, because oral candidiasis is commonplace in these patients over the last decade, research have indicated that PCP can be more common in TB smear-negative sufferers [47] [48] . The doubtful image of PCP incidence in Africa can be because of empirical remedy and prophylaxis the usage of cotrimoxazole in HIV/AIDS patients [49] .

Pneumonia is a significant economic burden on the healthcare system. In the developing world rates of PCP are lower, possibly due to poor survival, under diagnosis and prior infection with tuberculosis and other organisms [22] .

Sensitive and specific diagnostic tools are warranted in resource constrained setting and PCR presents a potential rapid sensitive diagnostic tool. Considering cost simplicity and efficacy we recommend TBO as the most practical diagnostic test for use in resource-constrained setting.

The data obtained from this study is an indication of the coinfection of fungi and pulmonary tuberculosis. The study signifies the need for an evaluation of the recurring pulmonary tuberculosis and prudent antifungal treatment based on the culture results rather than broad-spectrum antibiotics for cure. Induced sputum is more cost-effective; however, it is also of limited use because it requires staff training, patient preparation and dedicated rooms. A few studies have compared the value of induced sputum with expectorated sputum and they concluded that the difference in yield was negligible. The current study demonstrated that expectorated sputum can be used in resource-limited settings in sub-Saharan Africa for the routine diagnosis of PCP.

Pneumocystis jirovecii is fastidious and thus in vitro culture is difficult. As Pneumocystis remains a non-cultivable microorganism, traditional diagnostic methods rely on microscopic observations of the pathogen in respiratory specimen which is inherently non-specific.

Smear negative patients presenting with clinical symptoms of Tuberculosis the long run should undergo mycological testing before treatment. This necessitates investment in technical and infrastructure capabilities for the diagnosis of fungal illness such as Pneumocystis jirovecii.

This study was funded by the Kenya Medical Research Institute. I acknowledge my supervisors for their essential evaluation of my work and their consistent encouragement. Secondly, I acknowledge the Centre for Microbiology Research (CMR), KEMRI, for the supply of laboratory area and the work force. I additionally acknowledge the Coast general hospital, Mombasa for their help in acquiring samples from their hospital. Ultimately, my colleagues, friends and family for their consistent support.